6-substituted quinazolinone inhibitors

ABSTRACT

The present technology relates to compounds and compositions of Formulas I-III and methods using such compounds. The compounds and compositions described herein may be used in the treatment or prophylaxis of diseases associated with an alphavirus, for example, Venezuelan equine encephalitis virus (VEEV).

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/443,232, now U.S. Pat. No. 10,087,168, filed Feb. 27, 2017, which isa divisional of U.S. patent application Ser. No. 14/651,777, now U.S.Pat. No. 9,580,393, filed Jun. 12, 2015, which is the U.S. 371 NationalStage application of International Application No. PCT/US2013/075092,filed on Dec. 13, 2013, which claims priority to U.S. Provisional PatentApplication No. 61/737,005, filed Dec. 13, 2012, and U.S. ProvisionalPatent Application No. 61/853,740, filed on Apr. 11, 2013, the entiredisclosures of each of which are hereby incorporated by reference intheir entireties for any and all purposes.

GOVERNMENT FUNDING

This invention was made with government support under U54 HG005031, R⁰³MH087448, and U54 HG005034 awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD

The present technology is directed to compounds, compositions, andmethods to treat an alphavirus. The technology is especially suited totreat Venezuelan equine encephalitis virus (VEEV).

SUMMARY

The present technology is directed to compounds, compositions, andmethods to treat an alphavirus. The technology is especially suited totreat encephalitis alphaviruses. The compounds and compositionsdescribed herein may be used in the treatment or prophylaxis of diseasesthat include, for example, infections by alphaviruses.

In one aspect of the present technology, a compound of Formula I, II, orIII is provided:

stereoisomers thereof and pharmaceutically acceptable salts thereof,wherein W is CH or N; X₁ is an electron withdrawing group; X₂ ishydrogen or an electron withdrawing group; Y is O or S; R₁ is an alkylgroup, an aryl group, an aralkyl group, or a heteroaryl group; R₂ ishydrogen or alkyl; R₃ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; R₄ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; α is 0 or 1; B is CH, C-alkyl, O, or N; withthe provision that when B is O, R₄ is absent; Z is selected from thegroup consisting of

where R₅ is a hydrogen, alkyl, aryl, cycloalkyl, or non-aromaticheterocyclyl; A is CH, C-alkyl, or N; and n is 1, 2, 3, or 4. In someembodiments, the electron withdrawing group is a halogen, a nitro group,cyano group, an alkanoyl group, a carbamoyl group, an ester, a sulfonylgroup, a trialkyl ammonium group, or a trifluoromethyl group. In someembodiments, the electron withdrawing group is a halogen, a nitro group,a cyano group, or a trifluoromethyl group. In some embodiments, X₂ ishydrogen, a halogen, a nitro group, or a cyano group. In someembodiments, X₂ is hydrogen or a halogen. In some embodiments, X₂ ishydrogen. In some embodiments, R₂ is hydrogen.

In some embodiments, the compound is of Formulas I, II, or III and Y isO; R₁ is a heteroaryl group or a phenyl group, wherein the phenyl groupis of Formula IA:

where R₆ R₇, R₈, R₉ and R₁₀ are each independently hydrogen, halo,alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro; R₂ ishydrogen; α is 1; R₃ is a hydrogen or alkyl, R₄ and R₅ are eachindependently hydrogen, alkyl, aryl, cycloalkyl, non-aromaticheterocyclyl, alkanoyl, or carbamoyl; A is CH, C-alkyl or N; B is CH,C-alkyl, O, or N; with the provision that when B is O, R₄ is absent; andn is 1, 2, or 3.

In some embodiments, the compound is of Formulas I, II, or III and X₂ ishydrogen; Y is O; R₁ is an alkyl group, a heteroaryl group, or a phenylgroup wherein the phenyl group is of Formula IA where R₆ is hydrogen,methoxy, halo, alkanoyl, or nitro; R₇ and R₈ are each independentlyhydrogen, alkoxy, aryloxy, halo, alkanoyl, or nitro; R₉ and R₁₀ are eachindependently hydrogen; R₂ is hydrogen; α is 1; Z is selected from thegroup consisting of

where R₃ is hydrogen or alkyl; R₄ and R₅ are each independentlyhydrogen, alkyl, aryl, cycloalkyl, non-aromatic heterocyclyl, oralkanoyl; A is CH, C-alkyl, or N; B is CH, C-alkyl, or N; and n is 2 or3.

In some embodiments, the compound is of Formulas I, II, or III and X₁ isa halogen, a nitro group, or cyano group; X₂ is hydrogen; Y is O; R₁ isa methyl group, an ethyl group, or a phenyl group, wherein the phenylgroup is of Formula IA where R₆, R₇, and R₈ are each independentlyhydrogen, methoxy, halo, or nitro; R₉ and R₁₀ are each independentlyhydrogen; R₂ is hydrogen; α is 1; Z is selected from the groupconsisting of

where R₃ is hydrogen or alkyl; R₄ and R₅ are each independentlyhydrogen, alkyl, cycloalkyl, or non-aromatic heterocyclyl; A is CH,C-alkyl, or N; B is CH, C-alkyl, or N; and n is 2 or 3.

In some embodiments, R₆ is hydrogen. In some embodiments, Z is

R₃ is methyl or ethyl, R₄ is methyl, and R₅ is hydrogen. In someembodiments, Z is

and R₃ is hydrogen. In some embodiments, W is CH. In some embodiments,R₁ is a phenyl group of Formula IA where R₆ is hydrogen; R₇, and R₈ areeach independently hydrogen, methoxy, or halo; and R₉ and R₁₀ are eachindependently hydrogen.

In some embodiments, the compound is of Formula I.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀are each independently hydrogen, halo, alkoxy, alkanoyl, carbamoyl,cyano, trifluoromethyl, or nitro. In some embodiments, the compound isof Formulas II or III, R₁ is an alkyl group, an aralkyl group, aheteroaryl group, or a phenyl group, wherein the phenyl group is ofFormula IA where R₆, R₇, R₉, and R₁₀ are each independently hydrogen,halo, alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro; andR₈ is hydrogen, halo, substituted alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆, R₇, R₉, and R₁₀ areeach independently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; and R₈ is hydrogen, halo, cycloalkoxy,alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro. In someembodiments, the compound is of Formulas II or III; R₁ is an alkylgroup, an aralkyl group, a heteroaryl group, or a phenyl group, whereinthe phenyl group is of Formula IA where R₆, R₇, R₉, and R₁₀ are eachindependently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; and R₈ is hydrogen, halo, alkanoyl,carbamoyl, cyano, trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀are each independently hydrogen, halo, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl groupwherein the phenyl group is of Formula IA where R₆ is hydrogen, methoxy,halo, alkanoyl, or nitro; R₇ and R₈ are each independently hydrogen,alkoxy, aryloxy, halo, alkanoyl, or nitro; and R₉ and R₁₀ are eachindependently hydrogen. In some embodiments, the compound is of FormulasII or III; Y is O; R₁ is a heteroaryl group or a phenyl group, whereinthe phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀ are eachindependently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; R₂ is hydrogen; α is 1; R₄ and R₅ are eachindependently hydrogen, alkyl, aryl, cycloalkyl, non-aromaticheterocyclyl, alkanoyl, or carbamoyl; and B is CH, C-alkyl, O, or N;with the provision that when B is O, R₄ is absent.

In some embodiments, the compound is of Formulas II or III; X₂ ishydrogen; Y is O; R₁ is an alkyl group, a substituted or unsubstitutedbenzyl group, a heteroaryl group, or a phenyl group wherein the phenylgroup is of Formula IA where R₆ is hydrogen, methoxy, halo, alkanoyl, ornitro; R₇ and R₈ are each independently hydrogen, alkoxy, aryloxy, halo,alkanoyl, or nitro; R₉ and R₁₀ are each independently hydrogen; R₂ ishydrogen; α is 1; R₄ and R₅ are each independently hydrogen, alkyl,aryl, cycloalkyl, non-aromatic heterocyclyl, or alkanoyl; and B is CH,C-alkyl, or N.

In some embodiments, the compound is of Formulas II or III; X₁ is ahalogen, a nitro group, a trifluoromethyl group, or a cyano group; X₂ ishydrogen; Y is O; R₁ is a methyl group, an ethyl group, a benzyl group,or a phenyl group, wherein the phenyl group is of Formula IA where R₆,R₇, and R₈ are each independently hydrogen, methoxy, halo, or nitro; R₉and R₁₀ are each independently hydrogen; R₂ is hydrogen; α is 1; R₄ andR₅ are each independently hydrogen, alkyl, cycloalkyl, or non-aromaticheterocyclyl; B is CH, C-alkyl, or N.

In some embodiments, R₆ is hydrogen. In some embodiments, R₃ is methylor ethyl; and R₄ is methyl. In some embodimentsW is CH. In someembodiments, R₁ is a phenyl group of Formula IA where R₆ is hydrogen;R₇, and R₈ are each independently hydrogen, methoxy, or halo; R₉ and R₁₀are each independently hydrogen. In some embodiments, the compound is ofFormula II. In some embodiments, the compound is of Formula III.

In some embodiments, the compound is:2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,6-nitro-2-(piperazin-1-ylmethyl)-3-(thiophen-3-yl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(2-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(3-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(4-fluorophenyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-isopropylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,3-(2-fluorophenyl)-2-(morpholinomethyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-6-nitro-2-(piperidin-1-ylmethyl)quinazolin-4(3N)-one,3-(3-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,3-(4-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,2-((4-ethylpiperazin-1-yl)methyl)-6-iodo-3-phenylquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,6-nitro-3-phenyl-2-((tetrahydropyrimidin-1(2H)-yl)methyl)quinazolin-4(3H)-one,2-((1,4-diazepan-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-((methyl(3-(methylamino)propyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-(((2-(ethylamino)ethyl)(methyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,3-(4-methoxyphenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(3-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile,6-fluoro-2-((methyl(2-(methylamino)ethyl)amino)methyl)-3-phenylquinazolin-4(3N)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylpyrido[2,3-d]pyrimidin-4(3H)-one,6-nitro-3-phenyl-2-(piperidin-4-yl)quinazolin-4(3H)-one,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(E)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(E)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(E)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(E)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(Z)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(Z)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(Z)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(Z)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In some embodiments, the compound is(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(E)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(E)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(E)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(E)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In some embodiments, the compound is2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,6-nitro-2-(piperazin-1-ylmethyl)-3-(thiophen-3-yl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(2-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(3-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(4-fluorophenyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-isopropylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-(morpholinomethyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-6-nitro-2-(piperidin-1-ylmethyl)quinazolin-4(3H)-one,3-(3-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,3-(4-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-6-iodo-3-phenylquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,6-nitro-3-phenyl-2-((tetrahydropyrimidin-1(2H)-yl)methyl)quinazolin-4(3H)-one,2-((1,4-diazepan-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-((methyl(3-(methylamino)propyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-(((2-(ethylamino)ethyl)(methyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,3-(4-methoxyphenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3H)-one,3-(3-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile,6-fluoro-2-((methyl(2-(methylamino)ethyl)amino)methyl)-3-phenylquinazolin-4(3H)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylpyrido[2,3-d]pyrimidin-4(3H)-one,or 6-nitro-3-phenyl-2-(piperidin-4-yl)quinazolin-4(3H)-one.

In some embodiments, the compound is:(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(Z)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(Z)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(Z)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(Z)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In another aspect, the present technology provides a method comprisingadministering to a subject in need thereof an antiviral effective amountof a compound of Formulas I, II, or III. In the method, risk ofinfection by and/or transmission of an alphavirus by said subject isdecreased.

In another aspect, a composition is provided including a compound ofFormulas I, II, or III and a pharmaceutically acceptable carrier. In arelated aspect, a pharmaceutical composition is provided for treating aviral infection, where the composition includes an effective amount ofthe compound of Formulas I, II, or III. In some embodiments, the viralinfection comprises an alphavirus. In some embodiments, the viralinfection comprises an encephalitic alphavirus. In some embodiments, theviral infection comprises a Venezuelan equine encephalitis virus. Insome embodiments, the effective compound selectively treats the viralinfection. Thus, the instant present technology provides pharmaceuticalcompositions and medicaments comprising any of the compounds disclosedherein (e.g., compounds of Formulas I, II, or III) and apharmaceutically acceptable carrier or one or more excipients orfillers. The compositions may be used in the methods and treatmentsdescribed herein. Such compositions include a viral inhibitory effectiveamount of any compound as described herein, including but not limited toa compound of Formulas I, II, or III. In some embodiments, thepharmaceutical composition is packaged in unit dosage form. The unitdosage form is effective in preventing infection by, reducing symptomsassociated with, and/or reducing risk of transmission of an encephaliticalphavirus when administered to a subject in need thereof.

In one aspect, a method is provided for administering to a subject inneed thereof an antiviral effective amount of a compound of the presenttechnology. A subject in need thereof may be a patient suffering from orbelieved to be at risk of suffering from a disease associated with avirus, such as an encephalitic alphavirus. In some embodiments, thecompound is effective in the treatment of an alphavirus. In someembodiments, the compound is effective in the treatment of anencephalitic alphavirus. In some embodiments, the compound is effectivein the treatment of a Venezuelan equine encephalitis virus. In someembodiments, the compound selectively treats the viral infection. Insome embodiments, the risk of infection by and/or transmission of analphavirus by said subject is decreased. In any of these embodiments,the administration may include oral administration, parenteraladministration, or nasal administration. In any of these embodiments,the administration may include subcutaneous injections, intravenousinjections, intraperitoneal injections, or intramuscular injections. Inany of these embodiments, the administration may include oraladministration.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical stability of ML336 over 8 h in the presence ofa 5-fold concentration of dithiothreitol (“DTT”), according to oneembodiment.

DETAILED DESCRIPTION

In various aspects, the present technology provides novel compounds andmethods for treating a viral infection The compounds provided herein canbe formulated into pharmaceutical compositions and medicaments that areuseful in the disclosed methods. Also provided is the use of thecompounds in preparing pharmaceutical formulations and medicaments, theuse of the compounds in treating a viral infection.

The following terms are used throughout as defined below.

As used herein and in the appended claims, singular articles such as “a”and “an” and “the” and similar referents in the context of describingthe elements (especially in the context of the following claims) are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the claims unless otherwise stated. No language in the specificationshould be construed as indicating any non-claimed element as essential.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³²and S³⁵ are thus within the scope of the present technology. Proceduresfor inserting such labels into the compounds of the present technologywill be readily apparent to those skilled in the art based on thedisclosure herein.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup is substituted with one or more substituents, unless otherwisespecified. In some embodiments, a substituted group is substituted with1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groupsinclude: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy,aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups;carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines;alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones;sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and ring systemsin which a bond to a hydrogen atom is replaced with a bond to a carbonatom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

Alkyl groups include straight chain and branched chain alkyl groupshaving from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms.Examples of straight chain alkyl groups include groups such as methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octylgroups. Examples of branched alkyl groups include, but are not limitedto, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl,and 2,2-dimethylpropyl groups. Representative substituted alkyl groupsmay be substituted one or more times with substituents such as thoselisted above, and include without limitation haloalkyl (e.g.,trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups havingfrom 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocycliccycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In someembodiments, the cycloalkyl group has 3 to 8 ring members, whereas inother embodiments the number of ring carbon atoms range from 3 to 5, 3to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridgedcycloalkyl groups and fused rings, such as, but not limited to,bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substitutedcycloalkyl groups may be substituted one or more times with,non-hydrogen and non-carbon groups as defined above. However,substituted cycloalkyl groups also include rings that are substitutedwith straight or branched chain alkyl groups as defined above.Representative substituted cycloalkyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, 2,2-, 2,3-,2,4-2,5- or 2,6-disubstituted cyclohexyl groups, which may besubstituted with substituents such as those listed above.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, andtypically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups maybe substituted at the alkyl, the cycloalkyl or both the alkyl andcycloalkyl portions of the group. Representative substitutedcycloalkylalkyl groups may be mono-substituted or substituted more thanonce, such as, but not limited to, mono-, di- or tri-substituted withsubstituents such as those listed above.

Alkenyl groups include straight and branched chain alkyl groups asdefined above, except that at least one double bond exists between twocarbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group hasone, two, or three carbon-carbon double bonds. Examples include, but arenot limited to vinyl, allyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂,—C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, among others. Representativesubstituted alkenyl groups may be mono-substituted or substituted morethan once, such as, but not limited to, mono-, di- or tri-substitutedwith substituents such as those listed above.

Cycloalkenyl groups include cycloalkyl groups as defined above, havingat least one double bond between two carbon atoms. In some embodimentsthe cycloalkenyl group may have one, two or three double bonds but doesnot include aromatic compounds. Cycloalkenyl groups have from 4 to 14carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples ofcycloalkenyl groups include cyclohexenyl, cyclopentenyl,cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

Cycloalkenylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of the alkyl group is replaced with a bond to acycloalkenyl group as defined above. Substituted cycloalkenylalkylgroups may be substituted at the alkyl, the cycloalkenyl or both thealkyl and cycloalkenyl portions of the group. Representative substitutedcycloalkenylalkyl groups may be substituted one or more times withsubstituents such as those listed above.

Alkynyl groups include straight and branched chain alkyl groups asdefined above, except that at least one triple bond exists between twocarbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group hasone, two, or three carbon-carbon triple bonds. Examples include, but arenot limited to —C≡CH, —C≡CCH₃, —CH₂C≡CCH₃, —C≡CCH₂CH(CH₂CH₃)₂, amongothers. Representative substituted alkynyl groups may bemono-substituted or substituted more than once, such as, but not limitedto, mono-, di- or tri-substituted with substituents such as those listedabove.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups herein include monocyclic, bicyclic andtricyclic ring systems. Thus, aryl groups include, but are not limitedto, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl,anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In someembodiments, aryl groups contain 6-14 carbons, and in others from 6 to12 or even 6-10 carbon atoms in the ring portions of the groups. In someembodiments, the aryl groups are phenyl or naphthyl. Although the phrase“aryl groups” includes groups containing fused rings, such as fusedaromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, andthe like), it does not include aryl groups that have other groups, suchas alkyl or halo groups, bonded to one of the ring members. Rather,groups such as tolyl are referred to as substituted aryl groups.Representative substituted aryl groups may be mono-substituted orsubstituted more than once. For example, monosubstituted aryl groupsinclude, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenylor naphthyl groups, which may be substituted with substituents such asthose listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 16carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substitutedaralkyl groups may be substituted at the alkyl, the aryl or both thealkyl and aryl portions of the group. Representative aralkyl groupsinclude but are not limited to benzyl and phenethyl groups and fused(cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representativesubstituted aralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi-and tricyclic rings having 3 to 16 ring members, whereas other suchgroups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members.Heterocyclyl groups encompass aromatic, partially unsaturated andsaturated ring systems, such as, for example, imidazolyl, imidazolinyland imidazolidinyl groups. The phrase “heterocyclyl group” includesfused ring species including those comprising fused aromatic andnon-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. However, the phrase does notinclude heterocyclyl groups that have other groups, such as alkyl, oxoor halo groups, bonded to one of the ring members. Rather, these arereferred to as “substituted heterocyclyl groups”. Heterocyclyl groupsinclude, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl,tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl,imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl,thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl,thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane,dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl,pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl,dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl,isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl,benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl,benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl(azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl,xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl,quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl,pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl,dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl,tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl,tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl,tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, andtetrahydroquinolinyl groups. Representative substituted heterocyclylgroups may be mono-substituted or substituted more than once, such as,but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-,5-, or 6-substituted, or disubstituted with various substituents such asthose listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl(pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl(azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl,benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl,adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fusedring compounds in which all rings are aromatic such as indolyl groupsand include fused ring compounds in which only one of the rings isaromatic, such as 2,3-dihydro indolyl groups. Although the phrase“heteroaryl groups” includes fused ring compounds, the phrase does notinclude heteroaryl groups that have other groups bonded to one of thering members, such as alkyl groups. Rather, heteroaryl groups with suchsubstitution are referred to as “substituted heteroaryl groups.”Representative substituted heteroaryl groups may be substituted one ormore times with various substituents such as those listed above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheterocyclyl group as defined above. Substituted heterocyclylalkylgroups may be substituted at the alkyl, the heterocyclyl or both thealkyl and heterocyclyl portions of the group. Representativeheterocyclyl alkyl groups include, but are not limited to,morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl,pyridin-3-yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl.Representative substituted heterocyclylalkyl groups may be substitutedone or more times with substituents such as those listed above.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above. Substituted heteroaralkyl groups maybe substituted at the alkyl, the heteroaryl or both the alkyl andheteroaryl portions of the group. Representative substitutedheteroaralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the presenttechnology are designated by use of the suffix, “ene.” For example,divalent alkyl groups are alkylene groups, divalent aryl groups arearylene groups, divalent heteroaryl groups are divalent heteroarylenegroups, and so forth. Substituted groups having a single point ofattachment to the compound of the present technology are not referred tousing the “ene” designation. Thus, e.g., chloroethyl is not referred toherein as chloroethylene.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Representative substitutedalkoxy groups may be substituted one or more times with substituentssuch as those listed above.

The terms “alkanoyl” and “alkanoyloxy” as used herein can refer,respectively, to —C(O)-alkyl groups and —O—C(O)-alkyl groups, eachcontaining 2-5 carbon atoms.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, asubstituted or unsubstituted aryl group bonded to an oxygen atom and asubstituted or unsubstituted aralkyl group bonded to the oxygen atom atthe alkyl. Examples include but are not limited to phenoxy, naphthyloxy,and benzyloxy. Representative substituted aryloxy and arylalkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The term “carboxylate” as used herein refers to a —COOH group.

The term “ester” as used herein refers to —COOR³⁰ groups. R³⁰ is asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.

The term “amide” (or “amido”) includes C- and N-amide groups, i.e.,—C(O)NR³¹R³², and —NR³¹C(O)R³² groups, respectively. R³¹ and R³² areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl orheterocyclyl group as defined herein. Amido groups therefore include butare not limited to carbamoyl groups (—C(O)NH₂) and formamide groups(—NHC(O)H). In some embodiments, the amide is —NR³¹C(O)—(C₁₅ alkyl) andthe group is termed “carbonylamino,” and in others the amide is—NHC(O)-alkyl and the group is termed “alkanoylamino.”

The term “nitrile” or “cyano” as used herein refers to the —CN group.

Urethane groups include N- and O-urethane groups, i.e., —NR³³C(O)OR³⁴and —OC(O)NR³³R³⁴ groups, respectively. R³³ and R³⁴ are independently asubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R³³may also be H.

The term “amine” (or “amino”) as used herein refers to —NR³⁵R³⁶ groups,wherein R³⁵ and R³⁶ are independently hydrogen, or a substituted orunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,heterocyclylalkyl or heterocyclyl group as defined herein. In someembodiments, the amine is alkylamino, dialkylamino, arylamino, oralkylarylamino. In other embodiments, the amine is NH₂, methylamino,dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino,phenylamino, or benzylamino.

The term “sulfonamido” includes S- and N-sulfonamide groups, i.e.,—SO₂NR³⁸R³⁹ and —NR³⁸SO₂R³⁹ groups, respectively. R³⁸ and R³⁹ areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, orheterocyclyl group as defined herein. Sulfonamido groups thereforeinclude but are not limited to sulfamoyl groups (—SO₂NH₂). In someembodiments herein, the sulfonamido is —NHSO₂-alkyl and is referred toas the “alkylsulfonylamino” group.

The term “thiol” refers to —SH groups, while sulfides include —SR⁴⁰groups, sulfoxides include —S(O)R⁴¹ groups, sulfones include —SO₂R⁴²groups, and sulfonyls include —SO₂OR⁴³. R⁴⁰, R⁴¹, R⁴², and R⁴³ are eachindependently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl,alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group asdefined herein. In some embodiments the sulfide is an alkylthio group,—S-alkyl.

The term “urea” refers to —NR⁴⁴—C(O)—NR⁴⁵R⁴⁶ groups. R⁴⁴, R⁴⁵, and R⁴⁶groups are independently hydrogen, or a substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, orheterocyclylalkyl group as defined herein.

The term “amidine” refers to —C(NR⁴⁷)NR⁴⁸R⁴⁹ and —NR⁴⁷C(NR⁴⁸)R⁴⁹,wherein R⁴⁷, R⁴⁸, and R⁴⁹ are each independently hydrogen, or asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The term “guanidine” refers to —NR⁵⁰C(NR⁵¹)NR⁵²R⁵³, wherein R⁵⁰, R⁵¹,R⁵² and R⁵³ are each independently hydrogen, or a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein.

The term “enamine” refers to —C(R⁵⁴)═C(R⁵⁵)NR⁵⁶R⁵⁷ and—NR⁵⁴C(R⁵⁵)═C(R⁵⁶)R⁵⁷, wherein R⁵⁴, R⁵⁵, R⁵⁶ and R⁵⁷ are eachindependently hydrogen, a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “halogen” or “halo” as used herein refers to bromine, chlorine,fluorine, or iodine. In some embodiments, the halogen is fluorine. Inother embodiments, the halogen is chlorine or bromine.

The term “hydroxy” as used herein can refer to —OH or its ionized form,—O—.

The term “imide” refers to —C(O)NR⁵⁸C(O)R⁵⁹, wherein R⁵⁸ and R⁵⁹ areeach independently hydrogen, or a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “imine” refers to —CR⁶⁰(NR⁶¹) and —N(CR⁶⁰R⁶¹) groups, whereinR⁶⁰ and R⁶¹ are each independently hydrogen or a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein, with theproviso that R⁶⁰ and R⁶¹ are not both simultaneously hydrogen.

The term “nitro” as used herein refers to an —NO₂ group.

The term “trifluoromethyl” as used herein refers to —CF₃.

The term “trifluoromethoxy” as used herein refers to —OCF₃.

The phrase “selectively treats” as used herein will be understood bypersons of ordinary skill in the art and will vary to some extentdepending upon the context in which the phrase is used. If there areuses of the phrase which are not clear to persons of ordinary skill inthe art, given the context in which the phrase is used, the phrase atminimum refers to the compounds acting through a viral-specificmechanism of action, resulting in fewer off-target effects because thecompounds target the virus and not the host. The phrase may further bemodified as discussed herein, including Tables 2-9.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 atoms refers to groupshaving 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers togroups having 1, 2, 3, 4, or 5 atoms, and so forth.

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the present technology and include acid or baseaddition salts which retain the desired pharmacological activity and isnot biologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When the compound ofthe present technology has a basic group, such as, for example, an aminogroup, pharmaceutically acceptable salts can be formed with inorganicacids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuricacid, and phosphoric acid), organic acids (e.g. alginate, formic acid,acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid,tartaric acid, lactic acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (suchas aspartic acid and glutamic acid). When the compound of the presenttechnology has an acidic group, such as for example, a carboxylic acidgroup, it can form salts with metals, such as alkali and earth alkalimetals (e.g. Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines(e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, triethanolamine) or basic aminoacids (e.g. arginine, lysine and ornithine). Such salts can be preparedin situ during isolation and purification of the compounds or byseparately reacting the purified compound in its free base or free acidform with a suitable acid or base, respectively, and isolating the saltthus formed.

Those of skill in the art will appreciate that compounds of the presenttechnology may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or stereoisomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, stereochemical orgeometric isomeric forms, it should be understood that the presenttechnology encompasses any tautomeric, conformational isomeric,stereochemical and/or geometric isomeric forms of the compounds havingone or more of the utilities described herein, as well as mixtures ofthese various different forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The presence and concentrations of theisomeric forms will depend on the environment the compound is found inand may be different depending upon, for example, whether the compoundis a solid or is in an organic or aqueous solution. For example, inaqueous solution, quinazolinones may exhibit the following isomericforms, which are referred to as tautomers of each other:

Because of the limits of representing compounds by structural formulas,it is to be understood that all chemical formulas of the compoundsdescribed herein represent all tautomeric forms of compounds and arewithin the scope of the present technology.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these stereoisomersare all within the scope of the present technology.

The compounds of the present technology may exist as solvates,especially hydrates. Hydrates may form during manufacture of thecompounds or compositions comprising the compounds, or hydrates may formover time due to the hygroscopic nature of the compounds. Compounds ofthe present technology may exist as organic solvates as well, includingDMF, ether, and alcohol solvates among others. The identification andpreparation of any particular solvate is within the skill of theordinary artisan of synthetic organic or medicinal chemistry.

VEEV, a member of family Togaviridae, has been known to cause severeneurological diseases in humans and horses. Epidemics have occurredaffecting hundreds of thousands of people in the Americas for nearly acentury. An estimated 70,000 to 100,000 humans and similar numbers ofhorses infected with VEEV were reported during the last outbreak in1995. The disease in humans is characterized by fever, headache, andencephalitis to varying degrees and is sometimes fatal. The mortalityrate is below 1%; however, the neurological disease is present in up to14% of patients. The virus is usually transmitted via mosquito bite, butevidence supports viral transmission by aerosol. The modes oftransmission make VEEV infection very difficult to control duringoutbreaks. Thus, prophylaxis and efficacious treatments are critical tominimizing the impact of the transmissible disease on human and equines.

Currently, there are no FDA-approved vaccines or therapeutics for theencephalitic alphaviruses, which limit treatment to supportive care. TheUS Army has been developing vaccines for VEEV as they appreciate theimpact of the disease on soldiers as well as its potential use as abioweapon. The vaccines, which are comprised of attenuated live virus,are still in the investigational new drug (IND) stage and are onlyavailable through the Special Immunization Program at United State ArmyMedical Research Institute of Infectious Diseases (USAMRIID) forprotecting personnel working with the virus. A few other vaccinecandidates are in the IND stage, such as formalized killed TC-83 vaccineand the live attenuated V3526 vaccine. Again those vaccines have notbeen FDA-approved due to lack of efficacy and adverse effects seenduring clinical trials.

The alphaviruses, of which VEEV is a member, include other medicallyimportant viruses such as Eastern and Western equine encephalitisviruses (EEEV and WEEV respectively), Sindbis virus (SINV) andChikungunya (CHIK) viruses. VEE, EEE and CHIK viruses are listed asCenter for Disease Control (CDC) category B and C agents because oftheir potential military and bioterrorism threats. They are relativelyeasy to produce at high titers, are highly infectious by aerosol, andcan cause severely debilitating disease and death. Finally, VEEV,eastern (EEEV), western (WEEV) and Chikungunya (CHIK) viruses areclosely related phylogentically. Therefore, discovery of VEEV inhibitorswould be equally effective against closely related other encephaliticalphaviruses.

The present technology is directed to compounds, compositions, andmethods to treat an alphavirus. The technology is especially suited totreat an encephalitic alphavirus. The compounds and compositionsdescribed herein may be used in the treatment or prophylaxis of diseasesthat include, for example, Venezuelan equine encephalitis virus (VEEV).Methods of treatment include administering to a subject in need thereofa therapeutically effective amount of a compound or compositiondescribed herein. The compounds of the present technology can also beused in the treatment or prophylaxis of a disease state or maladycharacterized by or associated with an alphavirus. Generally,prophylactic or prophylaxis relates to a reduction in the likelihood ofthe patient developing a disorder such as VEEV infection or proceedingto a diagnosis state for the disorder. For example, the compounds of thepresent technology can be used prophylacticly as a measure designed topreserve health and prevent the spread or maturation of disease in apatient. It is also appreciated that the various modes of treatment orprevention of a disease such as an alphavirus infection can mean“substantial” treatment or prevention, which includes total but alsoless than total treatment or prevention, and in which some biologicallyor medically relevant result is achieved. Furthermore, treatment ortreating as well as alleviating can refer to therapeutic treatment andprophylactic or preventative measures in which the object is to prevent,slow down (lessen) a disease state, condition or malady. For example, asubject can be successfully treated for an alphavirus infection if,after receiving through administration an effective or therapeuticamount of one or more compounds described herein, the subject showsobservable and/or measurable reduction in or absence of one or moresigns and symptoms of the particular disease. The present technologyalso provides for methods of administering one or more compounds of thepresent technology to a patient in an effective amount for the treatmentor prophylaxis of a disease such as, for example, an alphavirusinfection.

While not wishing to be bound by theory, it is believed that thecompounds and compositions disclosed herein act through a post-entry,viral-specific, mechanism of action by inhibiting viral replicationthrough the nsP2 helicase, resulting in the prevention or treatment ofdiseases related to an encephalitic alphavirus. “Virus specific” meansthe compounds do not use host cellular machinery to inhibit virus. Thus,there are fewer off-target effects because the compounds target thevirus and not host.

In one aspect of the present technology, a compound of Formula I, II, orIII is provided:

stereoisomers thereof and pharmaceutically acceptable salts thereof,wherein W is CH or N; X₁ is an electron withdrawing group; X₂ ishydrogen or an electron withdrawing group; Y is O or S; R₁ is an alkylgroup, an aryl group, an aralkyl group, or a heteroaryl group; R₂ ishydrogen or alkyl; R₃ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; R₄ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; α is 0 or 1; B is CH, C-alkyl, O, or N; withthe provision that when B is O, R₄ is absent; Z is selected from thegroup consisting of

where R₅ is a hydrogen, alkyl, aryl, cycloalkyl, or non-aromaticheterocyclyl; A is CH, C-alkyl, or N; and n is 1, 2, 3, or 4. If both X₁and X₂ are an electron withdrawing group, X₁ and X₂ may be differentelectron withdrawing groups or X₁ and X₂ may each independently bear thesame electron withdrawing group. In some embodiments, the electronwithdrawing group is a halogen, a nitro group, cyano group, an alkanoylgroup, a carbamoyl group, an ester, a sulfonyl group, a trialkylammonium group, or a trifluoromethyl group. In some embodiments, theelectron withdrawing group is a halogen, a nitro group, a cyano group,or a trifluoromethyl group. In some embodiments, X₂ is hydrogen, ahalogen, a nitro group, or a cyano group. In some embodiments, X₂ ishydrogen or a halogen. In some embodiments, X₂ is hydrogen. In someembodiments, R₂ is hydrogen.

In some embodiments, the compound is of Formulas I, II, or III and Y isO; R₁ is a heteroaryl group or a phenyl group, wherein the phenyl groupis of Formula IA:

where R₆ R₇, R₈, R₉ and R₁₀ are each independently hydrogen, halo,alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro; R₂ ishydrogen; α is 1; R₃ is a hydrogen or alkyl, R₄ and R₅ are eachindependently hydrogen, alkyl, aryl, cycloalkyl, non-aromaticheterocyclyl, alkanoyl, or carbamoyl; A is CH, C-alkyl or N; B is CH,C-alkyl, O, or N; with the provision that when B is O, R₄ is absent; andn is 1, 2, or 3.

In some embodiments, the compound is of Formulas I, II, or III and X₂ ishydrogen; Y is O; R₁ is an alkyl group, a heteroaryl group, or a phenylgroup wherein the phenyl group is of Formula IA where R₆ is hydrogen,methoxy, halo, alkanoyl, or nitro; R₇ and R₈ are each independentlyhydrogen, alkoxy, aryloxy, halo, alkanoyl, or nitro; R₉ and R₁₀ are eachindependently hydrogen; R₂ is hydrogen; α is 1; Z is selected from thegroup consisting of

where R₃ is hydrogen or alkyl; R₄ and R₅ are each independentlyhydrogen, alkyl, aryl, cycloalkyl, non-aromatic heterocyclyl, oralkanoyl; A is CH, C-alkyl, or N; B is CH, C-alkyl, or N; and n is 2 or3.

In some embodiments, the compound is of Formulas I, II, or III and X₁ isa halogen, a nitro group, or cyano group; X₂ is hydrogen; Y is O; R₁ isa methyl group, an ethyl group, or a phenyl group, wherein the phenylgroup is of Formula IA where R₆, R₇, and R₈ are each independentlyhydrogen, methoxy, halo, or nitro; R₉ and R₁₀ are each independentlyhydrogen; R₂ is hydrogen; α is 1; Z is selected from the groupconsisting of

where R₃ is hydrogen or alkyl; R₄ and R₅ are each independentlyhydrogen, alkyl, cycloalkyl, or non-aromatic heterocyclyl; A is CH,C-alkyl, or N; B is CH, C-alkyl, or N; and n is 2 or 3.

In some embodiments, R₆ is hydrogen. In some embodiments, Z is

R₃ is methyl or ethyl, R₄ is methyl, and R₅ is hydrogen. In someembodiments, Z is

and R₃ is hydrogen. In some embodiments, W is CH. In some embodiments,R₁ is a phenyl group of Formula IA where R₆ is hydrogen; R₇, and R₈ areeach independently hydrogen, methoxy, or halo; and R₉ and R₁₀ are eachindependently hydrogen.

In some embodiments, the compound is of Formula I.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀are each independently hydrogen, halo, alkoxy, alkanoyl, carbamoyl,cyano, trifluoromethyl, or nitro. In some embodiments, the compound isof Formulas II or III, R₁ is an alkyl group, an aralkyl group, aheteroaryl group, or a phenyl group, wherein the phenyl group is ofFormula IA where R₆, R₇, R₉, and R₁₀ are each independently hydrogen,halo, alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro; andR₈ is hydrogen, halo, substituted alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆, R₇, R₉, and R₁₀ areeach independently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; and R₈ is hydrogen, halo, cycloalkoxy,alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro. In someembodiments, the compound is of Formulas II or III; R₁ is an alkylgroup, an aralkyl group, a heteroaryl group, or a phenyl group, whereinthe phenyl group is of Formula IA where R₆, R₇, R₉, and R₁₀ are eachindependently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; and R₈ is hydrogen, halo, alkanoyl,carbamoyl, cyano, trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl group,wherein the phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀are each independently hydrogen, halo, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro.

In some embodiments, the compound is of Formulas II or III; R₁ is analkyl group, an aralkyl group, a heteroaryl group, or a phenyl groupwherein the phenyl group is of Formula IA where R₆ is hydrogen, methoxy,halo, alkanoyl, or nitro; R₇ and R₈ are each independently hydrogen,alkoxy, aryloxy, halo, alkanoyl, or nitro; and R₉ and R₁₀ are eachindependently hydrogen. In some embodiments, the compound is of FormulasII or III; Y is O; R₁ is a heteroaryl group or a phenyl group, whereinthe phenyl group is of Formula IA where R₆ R₇, R₈, R₉ and R₁₀ are eachindependently hydrogen, halo, alkoxy, alkanoyl, carbamoyl, cyano,trifluoromethyl, or nitro; R₂ is hydrogen; α is 1; R₄ and R₅ are eachindependently hydrogen, alkyl, aryl, cycloalkyl, non-aromaticheterocyclyl, alkanoyl, or carbamoyl; and B is CH, C-alkyl, O, or N;with the provision that when B is O, R₄ is absent.

In some embodiments, the compound is of Formulas II or III; X₂ ishydrogen; Y is O; R₁ is an alkyl group, a substituted or unsubstitutedbenzyl group, a heteroaryl group, or a phenyl group wherein the phenylgroup is of Formula IA where R₆ is hydrogen, methoxy, halo, alkanoyl, ornitro; R₇ and R₈ are each independently hydrogen, alkoxy, aryloxy, halo,alkanoyl, or nitro; R₉ and R₁₀ are each independently hydrogen; R₂ ishydrogen; α is 1; R₄ and R₅ are each independently hydrogen, alkyl,aryl, cycloalkyl, non-aromatic heterocyclyl, or alkanoyl; and B is CH,C-alkyl, or N.

In some embodiments, the compound is of Formulas II or III; X₁ is ahalogen, a nitro group, a trifluoromethyl group, or a cyano group; X₂ ishydrogen; Y is O; R₁ is a methyl group, an ethyl group, a benzyl group,or a phenyl group, wherein the phenyl group is of Formula IA where R₆,R₇, and R₈ are each independently hydrogen, methoxy, halo, or nitro; R₉and R₁₀ are each independently hydrogen; R₂ is hydrogen; α is 1; R₄ andR₅ are each independently hydrogen, alkyl, cycloalkyl, or non-aromaticheterocyclyl; B is CH, C-alkyl, or N.

In some embodiments, R₆ is hydrogen. In some embodiments, R₃ is methylor ethyl; and R₄ is methyl. In some embodiments, W is CH. In someembodiments, R₁ is a phenyl group of Formula IA where R₆ is hydrogen;R₇, and R₈ are each independently hydrogen, methoxy, or halo; R₉ and R₁₀are each independently hydrogen. In some embodiments, the compound is ofFormula II. In some embodiments, the compound is of Formula III.

In some embodiments, the compound is:2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,6-nitro-2-(piperazin-1-ylmethyl)-3-(thiophen-3-yl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(2-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(3-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(4-fluorophenyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-isopropylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-(morpholinomethyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-6-nitro-2-(piperidin-1-ylmethyl)quinazolin-4(3N)-one,3-(3-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,3-(4-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,2-((4-ethylpiperazin-1-yl)methyl)-6-iodo-3-phenylquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,6-nitro-3-phenyl-2-((tetrahydropyrimidin-1(2H)-yl)methyl)quinazolin-4(3H)-one,2-((1,4-diazepan-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-((methyl(3-(methylamino)propyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-(((2-(ethylamino)ethyl)(methyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,3-(4-methoxyphenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(3-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile,6-fluoro-2-((methyl(2-(methylamino)ethyl)amino)methyl)-3-phenylquinazolin-4(3N)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylpyrido[2,3-d]pyrimidin-4(3H)-one,6-nitro-3-phenyl-2-(piperidin-4-yl)quinazolin-4(3H)-one,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(E)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(E)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(E)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(E)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(Z)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(Z)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(Z)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(Z)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In some embodiments, the compound is(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(E)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(E)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(E)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(E)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In some embodiments, the compound is2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one,6-nitro-2-(piperazin-1-ylmethyl)-3-(thiophen-3-yl)quinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3N)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(2-fluorophenyl)-6-nitroquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(3-fluorophenyl)-6-nitroquinazolin-4(3N)-one,2-((4-ethylpiperazin-1-yl)methyl)-3-(4-fluorophenyl)-6-nitroquinazolin-4(3H)-one,3-(2-fluorophenyl)-2-((4-isopropylpiperazin-1-yl)methyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,3-(2-fluorophenyl)-2-(morpholinomethyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-6-nitro-2-(piperidin-1-ylmethyl)quinazolin-4(3N)-one,3-(3-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,3-(4-methoxyphenyl)-6-nitro-2-(piperazin-1-ylmethyl)quinazolin-4(3N)-one,2-((4-ethylpiperazin-1-yl)methyl)-6-iodo-3-phenylquinazolin-4(3H)-one,2-((4-ethylpiperazin-1-yl)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,6-nitro-3-phenyl-2-((tetrahydropyrimidin-1(2H)-yl)methyl)quinazolin-4(3H)-one,2-((1,4-diazepan-1-yl)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-((methyl(3-(methylamino)propyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,2-(((2-(ethylamino)ethyl)(methyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3N)-one,3-(4-methoxyphenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(2-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitroquinazolin-4(3N)-one,3-(3-fluorophenyl)-2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile,6-fluoro-2-((methyl(2-(methylamino)ethyl)amino)methyl)-3-phenylquinazolin-4(3N)-one,2-((methyl(2-(methylamino)ethyl)amino)methyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile,2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-3-phenylpyrido[2,3-d]pyrimidin-4(3N)-one,or 6-nitro-3-phenyl-2-(piperidin-4-yl)quinazolin-4(3H)-one.

In some embodiments, the compound is:(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(Z)-4-chloro-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(Z)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(Z)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(Z)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,or(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.

In another aspect, the present technology provides a method comprisingadministering to a subject in need thereof an antiviral effective amountof a compound of Formulas I, II, or III. In the method, risk ofinfection by and/or transmission of an alphavirus by said subject isdecreased.

“Effective amount” refers to the amount of a compound or compositionrequired to produce a desired effect. One example of an effective amountincludes amounts or dosages that yield acceptable toxicity andbioavailability levels for therapeutic (pharmaceutical) use including,but not limited to, the treatment or prophylaxis of an encephaliticalphavirus. Another example of an effective amount includes amounts ordosages that are capable of reducing symptoms associated with anencephalitic alphavirus, such as, for example, fever, headache, andencephalitis.

As used herein, a “subject” or “patient” is a mammal, such as a cat,dog, rodent or primate. Typically the subject is a human, and,preferably, a human suspected of having a disease associated with analphavirus. The term “subject” and “patient” can be usedinterchangeably.

In another aspect, a composition is provided including a compound ofFormulas I, II, or III and a pharmaceutically acceptable carrier. In arelated aspect, a pharmaceutical composition is provided for treating aviral infection, where the composition includes an effective amount ofthe compound of Formulas I, II, or III. In some embodiments, the viralinfection comprises an alphavirus. In some embodiments, the viralinfection comprises an encephalitic alphavirus. In some embodiments, theviral infection comprises a Venezuelan equine encephalitis virus. Insome embodiments, the effective compound selectively treats the viralinfection. Thus, the instant present technology provides pharmaceuticalcompositions and medicaments comprising any of the compounds disclosedherein (e.g., compounds of Formulas I, II, or III) and apharmaceutically acceptable carrier or one or more excipients orfillers. The compositions may be used in the methods and treatmentsdescribed herein. Such compositions include a viral inhibitory effectiveamount of any compound as described herein, including but not limited toa compound of Formulas I, II, or III. In some embodiments, thepharmaceutical composition is packaged in unit dosage form. The unitdosage form is effective in preventing infection by, reducing symptomsassociated with, and/or reducing risk of transmission of an encephaliticalphavirus when administered to a subject in need thereof.

The pharmaceutical compositions may be prepared by mixing one or morecompounds of the present technology, pharmaceutically acceptable saltsthereof, stereoisomers thereof, tautomers thereof, or solvates thereof,with pharmaceutically acceptable carriers, excipients, binders, diluentsor the like to prevent and treat disorders associated with the effectsof increased plasma and/or hepatic lipid levels. The compounds andcompositions described herein may be used to prepare formulations andmedicaments that prevent or treat a variety of disorders associated withan encephalitic alphavirus. Such compositions can be in the form of, forexample, granules, powders, tablets, capsules, syrup, suppositories,injections, emulsions, elixirs, suspensions or solutions. The instantcompositions can be formulated for various routes of administration, forexample, by oral, parenteral, topical, rectal, nasal, vaginaladministration, or via implanted reservoir. Parenteral or systemicadministration includes, but is not limited to, subcutaneous,intravenous, intraperitoneal, and intramuscular, injections. Thefollowing dosage forms are given by way of example and should not beconstrued as limiting the instant present technology.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant present technology, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Typically, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Compounds of the present technology may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. The carriers and stabilizers varywith the requirements of the particular compound, but typically includenonionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant)aerosols are typically used for delivery of compounds of the presenttechnology by inhalation.

Dosage forms for the topical (including buccal and sublingual) ortransdermal administration of compounds of the present technologyinclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, and patches. The active component may be mixed under sterileconditions with a pharmaceutically-acceptable carrier or excipient, andwith any preservatives, or buffers, which may be required. Powders andsprays can be prepared, for example, with excipients such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. The ointments, pastes, creamsand gels may also contain excipients such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Absorption enhancers can also be used toincrease the flux of the compounds of the present technology across theskin. The rate of such flux can be controlled by either providing a ratecontrolling membrane (e.g., as part of a transdermal patch) ordispersing the compound in a polymer matrix or gel.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instant presenttechnology. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

The formulations of the present technology may be designed to beshort-acting, fast-releasing, long-acting, and sustained-releasing asdescribed below. Thus, the pharmaceutical formulations may also beformulated for controlled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant present technology.

Those skilled in the art are readily able to determine an effectiveamount by simply administering a compound of the present technology to apatient in increasing amounts until the elevated plasma or elevatedwhite blood cell count or hepatic cholesterol or triglycerides orprogression of the disease state is decreased or stopped. Theprogression of the disease state can be assessed using in vivo imaging,as described, or by taking a tissue sample from a patient and observingthe target of interest therein. The compounds of the present technologycan be administered to a patient at dosage levels in the range of about0.1 to about 1,000 mg per day. For a normal human adult having a bodyweight of about 70 kg, a dosage in the range of about 0.01 to about 100mg per kg of body weight per day is sufficient. The specific dosageused, however, can vary or may be adjusted as considered appropriate bythose of ordinary skill in the art. For example, the dosage can dependon a number of factors including the requirements of the patient, theseverity of the condition being treated and the pharmacological activityof the compound being used. The determination of optimum dosages for aparticular patient is well known to those skilled in the art.

Various assays and model systems can be readily employed to determinethe therapeutic effectiveness of the viral treatment according to thepresent technology.

Effectiveness of the compositions and methods of the present technologymay also be demonstrated by a decrease in the symptoms of anencephalitic alphavirus, such as, for example, fever, headache, andencephalitis.

For each of the indicated conditions described herein, test subjectswill exhibit a 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%,or 95% or greater, reduction, in one or more symptom(s) caused by, orassociated with, viral infection in the subject, compared toplacebo-treated or other suitable control subjects.

The compounds of the present technology can also be administered to apatient along with other conventional therapeutic agents that may beuseful in the treatment or prophylaxis of viral infection. In oneaspect, a method is provided for administering to a subject in needthereof an antiviral effective amount of a compound of the presenttechnology. A subject in need thereof may be a patient suffering from orbelieved to be at risk of suffering from a disease associated with avirus, such as an encephalitic alphavirus. In some embodiments, thecompound is effective in the treatment of an alphavirus. In someembodiments, the compound is effective in the treatment of anencephalitic alphavirus. In some embodiments, the compound is effectivein the treatment of a Venezuelan equine encephalitis virus. In someembodiments, the compound selectively treats the viral infection. Insome embodiments, the risk of infection by and/or transmission of analphavirus by said subject is decreased. In any of these embodiments,the administration may include oral administration, parenteraladministration, or nasal administration. In any of these embodiments,the administration may include subcutaneous injections, intravenousinjections, intraperitoneal injections, or intramuscular injections. Inany of these embodiments, the administration may include oraladministration. The methods of the present technology can also compriseadministering, either sequentially or in combination with one or morecompounds of the present technology, a conventional therapeutic agent inan amount that can potentially or synergistically be effective for thetreatment or prophylaxis of encephalitic alphavirus. Exemplarytherapeutic agents for use in combination therapies with one or morecompounds of the present technology include, but are not limited to,other antiviral therapeutics, antibiotics, and anti-inflammatory drugs.

In one aspect, a compound of the present technology is administered to apatient in an amount or dosage suitable for therapeutic use. Generally,a unit dosage comprising a compound of the present technology will varydepending on patient considerations. Such considerations include, forexample, age, protocol, condition, sex, extent of disease,contraindications, concomitant therapies and the like. An exemplary unitdosage based on these considerations can also be adjusted or modified bya physician skilled in the art. For example, a unit dosage for a patientcomprising a compound of the present technology can vary from 1×10⁻⁴g/kg to 1 g/kg, preferably, 1×10⁻³ g/kg to 1.0 g/kg. Dosage of acompound of the present technology can also vary from 0.01 mg/kg to 100mg/kg or, preferably, from 0.1 mg/kg to 10 mg/kg.

A compound of the present technology can also be modified, for example,by the covalent attachment of an organic moiety or conjugate to improvepharmacokinetic properties, toxicity or bioavailability (e.g., increasedin vivo half-life). The conjugate can be a linear or branchedhydrophilic polymeric group, fatty acid group or fatty acid ester group.A polymeric group can comprise a molecular weight that can be adjustedby one of ordinary skill in the art to improve, for example,pharmacokinetic properties, toxicity or bioavailability. Exemplaryconjugates can include a polyalkane glycol (e.g., polyethylene glycol(PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acidpolymer or polyvinyl pyrolidone and a fatty acid or fatty acid estergroup, each of which can independently comprise from about eight toabout seventy carbon atoms. Conjugates for use with a compound of thepresent technology can also serve as linkers to, for example, anysuitable substituents or groups, radiolabels (marker or tags), halogens,proteins, enzymes, polypeptides, other therapeutic agents (for example,a pharmaceutical or drug), nucleosides, dyes, oligonucleotides, lipids,phospholipids and/or liposomes. In one aspect, conjugates can includepolyethylene amine (PEI), polyglycine, hybrids of PEI and polyglycine,polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). Aconjugate can also link a compound of the present technology to, forexample, a label (fluorescent or luminescent) or marker (radionuclide,radioisotope and/or isotope) to comprise a probe of the presenttechnology. Conjugates for use with a compound of the present technologycan, in one aspect, improve in vivo half-life. Other exemplaryconjugates for use with a compound of the present technology as well asapplications thereof and related techniques include those generallydescribed by U.S. Pat. No. 5,672,662, which is hereby incorporated byreference herein.

In another aspect, the present technology provides methods ofidentifying a target of interest including contacting the target ofinterest with a detectable or imaging effective quantity of a labeledcompound of the present technology. A detectable or imaging effectivequantity is a quantity of a labeled compound of the present technologynecessary to be detected by the detection method chosen. For example, adetectable quantity can be an administered amount sufficient to enabledetection of binding of the labeled compound to a target of interestincluding, but not limited to, VEEV nsP2 helicase. Suitable labels areknown by those skilled in the art and can include, for example,radioisotopes, radionuclides, isotopes, fluorescent groups, biotin (inconjunction with streptavidin complexation), and chemoluminescentgroups. Upon binding of the labeled compound to the target of interest,the target may be isolated, purified and further characterized such asby determining the amino acid sequence.

The terms “associated” and/or “binding” can mean a chemical or physicalinteraction, for example, between a compound of the present technologyand a target of interest. Examples of associations or interactionsinclude covalent bonds, ionic bonds, hydrophilic-hydrophilicinteractions, hydrophobic-hydrophobic interactions and complexes.Associated can also refer generally to “binding” or “affinity” as eachcan be used to describe various chemical or physical interactions.Measuring binding or affinity is also routine to those skilled in theart. For example, compounds of the present technology can bind to orinteract with a target of interest or precursors, portions, fragmentsand peptides thereof and/or their deposits.

The examples herein are provided to illustrate advantages of the presenttechnology and to further assist a person of ordinary skill in the artwith preparing or using the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, metabolites, prodrugs,racemic mixtures or tautomeric forms thereof. The examples herein arealso presented in order to more fully illustrate the preferred aspectsof the present technology. The examples should in no way be construed aslimiting the scope of the present technology, as defined by the appendedclaims. The examples can include or incorporate any of the variations,aspects or aspects of the present technology described above. Thevariations, aspects or aspects described above may also further eachinclude or incorporate the variations of any or all other variations,aspects or aspects of the present technology.

EXAMPLES

General Synthetic and Analytical Details:

¹H and ¹³C NMR spectra were recorded on a Bruker AM 400 spectrometer(operating at 400 and 101 MHz respectively) or a Bruker AVIIIspectrometer (operating at 500 and 126 MHz respectively) in CDCl₃ with0.03% TMS as an internal standard or DMSO-d₆. The chemical shifts (6)reported are given in parts per million (ppm) and the coupling constants(J) are in Hertz (Hz). The spin multiplicities are reported ass=singlet, bs=broad singlet, d=doublet, t=triplet, q=quartet, dd=doubletof doublet and m=multiplet. The LCMS analysis was performed on anAgilent 1200 RRL chromatograph with photodiode array UV detection and anAgilent 6224 TOF mass spectrometer. The chromatographic method utilizedthe following parameters: a Waters Acquity BEH C-18 2.1×50 mm, 1.7 umcolumn; UV detection wavelength=214 nm; flow rate=0.4 ml/min;gradient=5-100% acetonitrile over 3 minutes with a hold of 0.8 minutesat 100% acetonitrile; the aqueous mobile phase contained 0.15% ammoniumhydroxide (v/v). The mass spectrometer utilized the followingparameters: an Agilent multimode source which simultaneously acquiresESI+/APCI+; a reference mass solution consisting of purine andhexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-upsolvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to theLC flow prior to the source to assist ionization. Melting points weredetermined on a Stanford Research Systems OptiMelt apparatus.

Synthesis of(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide(“ML336”)

A general synthetic scheme is shown in Scheme 1, followed by a detaileddescription of the synthesis of ML336.

Synthesis of 2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one

2-amino-5-nitro-benzoic acid (6.718 g, 36.9 mmol, 1 eq.) was placedunder nitrogen and dissolved in CH₂Cl₂ (100 mL). After addition oftriethylamine (5.9 mL, 42.3 mmol, 1.15 eq), the mixture was lowered to0° C. in an ice bath and a solution of chloroacetyl chloride (3.2 mL,40.2 mmol, 1.1 eq) in CH₂Cl₂ (50 mL) was slowly added. The reactionmixture was stirred at 0° C. for 1 hour, then at rt for 1 additionalhour. The solvent was removed in vacuo and water was added to theremaining solid. The solid was filtered and rinsed with water (3×20 mL),followed by 5% Et₂O/Hexanes (3×30 mL) to give2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (8.87 g, 100%)along with residual triethylamine as a pale, yellow solid. The productwas used in the following step without further purification. ¹H NMR (400MHz, acetone-d₆) δ 8.96-8.91 (m, 2H), 8.48 (dd, J=9.3, 2.8 Hz, 1H), 4.44(s, 2H).

2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one

To a microwave vial was added2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (1.47 g, 6.12mmol, 1 eq) under Ar, and the solid was dissolved in acetonitrile (13mL). Phosphorus oxychloride (1.15 mL, 12.34 mmol, 2 eq) was added,followed by the addition of solution of aniline (0.73 mL, 8.00 mmol, 1.3eq) in acetonitrile (4 mL). The mixture was heated in a MW reactor at150° C. for 15 min. The reaction mixture was transferred to a largerflask and slowly quenched with saturated aq. NaHCO₃(20 mL). Theprecipitate was filtered and rinsed with water (3×20 mL) to give2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.24 g, 64%) as aburnt-orange solid. ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J=2.6 Hz, 1H),8.59 (dd, J=8.9, 2.6 Hz, 1H), 7.91 (d, J=9.0 Hz, 1H), 7.65-7.58 (m, 3H),7.39-7.35 (m, 2H), 4.28 (s, 2H).

tert-butyl-methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate

2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.40 g, 4.43mmol, 1 eq) was dissolved in acetonitrile (18 mL). Potassium carbonate(1.226 g, 8.87 mmol, 2 eq), tert-butylmethyl(2-(methylamino)ethyl)carbamate (ChemBridge, 1.18 g, 6.25 mmol,1.4 eq), and potassium iodide (0.280 g, 1.687 mmol, 0.4 eq) were addedsequentially, and the mixture was heated in a MW reactor at 80° C. for 5min. The crude reaction mixture was adsorbed onto Celite®, and theproduct was purified (2×) by flash chromatography (CombiFlash, 40 gsilica, 0-10% MeOH/CH₂Cl₂, followed by CombiFlash, 80 g silica, 0-80%EtOAc/Hexanes) to givetert-butyl-methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate(0.72 g, 35%) as a pale orange solid. ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d,J=2.6 Hz, 1H), 8.56 (dd, J=9.0, 2.7 Hz, 1H), 7.89 (d, J=9.0 Hz, 1H),7.60-7.52 (m, 3H), 7.32-7.27 (m, 2H), 3.36 (s, 2H), 3.14 (br d, J=19.4Hz, 2H), 2.75 (br s, 3H), 2.48 (br s, 2H), 2.20 (s, 3H), 1.39 (br s,9H).

(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide(“ML336”)

To a stirred solution of tert-butylmethyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate(678 mg, 1.45 mmol) in CH₂Cl₂ (20 mL), TFA (9.5 mL, 124 mmol) was slowlyadded, and the resulting mixture was stirred at rt for 45 min. Water (40mL) and CH₂Cl₂ (40 mL) were then added, and the mixture was adjusted topH 10 using saturated aq. Na₂CO₃ (40 mL). The organic phase wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×40 mL). Thecombined organic phase was concentrated and purified by flashchromatography (CombiFlash, 40 g silica, 0-5% MeOH/CH₂Cl₂) to give theproduct (265 mg, 49%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃) δ10.99 (s, 1H), 9.15 (d, J=2.8 Hz, 1H), 8.15 (dd, J=8.8, 2.8 Hz, 1H),7.65-7.60 (m, 2H), 7.38-7.33 (m, 2H), 7.12 (tt, J=7.3, 1.2 Hz, 1H), 6.80(d, J=8.8 Hz, 1H), 3.47 (t, J=5.7 Hz, 2H), 3.28 (s, 3H), 3.13 (s, 2H),2.69 (t, J=5.7 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 162.63,156.42, 153.97, 142.79, 138.19, 129.09, 127.64, 126.35, 126.16, 124.19,123.75, 120.19, 55.12, 51.82, 49.66, 45.27, 36.88. LCMS retention time:3.206 min, purity at 214 nm=99.2%. HRMS m/z calculated for C₁₉H₂₂N₅O₃[M⁺+H] 368.1723, found 368.1718. Pale yellow needles, mp 168-173° C.(recrystallized from CH₂Cl₂).

While the product was initially logged as2-((methyl(2-(methylamino)ethyl)amino)methyl)-6-nitro-3-phenylquinazolin-4(3H)-one,upon inspection of the ¹H NMR and ¹³C NMR it was clear that(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamidewas formed (265 mg, 49%) as a pale yellow solid. Particularly tellingwas the singlet at δ 10.99 corresponding to 1 hydrogen atom. While thiswas the proton bonded to the nitrogen, it was at a significantly highershift than would be expected for a secondary amine. Such a shift isnormally seen in amide protons where the amide nitrogen is furtherconjugated with an aromatic ring. Similarly, the E configurationappeared to be the correct isomer due not only to allylic strainconsiderations, but also to the absence of higher order splitting of theproton ortho to the aromatic carbon bearing the sp² amidine nitrogen andthe protons of the methyl group on the sp³ amidine nitrogen, as wouldlikely be observed with the Z isomer. Due to this and otherconsiderations known to those of ordinary skill in the art, it wasapparent that(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamidewas the product.

The structural connectivity of ML336 was further confirmed using 2Dproton-carbon HSQC and HMBC correlations, in conjunction with the above¹H and ¹³C NMR shifts (Diagrams 1 and 2).

Diagram 1. Correlation assignments

Position ¹H NMR σ ¹³C NMR σ A 7.12 (tt, J = 7.3, 1.2 Hz, 1H) 124.19 B7.38-7.33 (m, 2H) 129.09 C 7.65-7.60 (m, 2H) 120.19 D 138.19 E 10.99 (s,1H) F 162.63 G 126.16 H 9.15 (d, J = 2.8 Hz, 1H) 127.64 I 142.79 J 8.15(dd, J = 8.8, 2.8 Hz, 1H) 126.35 K 6.80 (d, J = 8.8 Hz, 1H) 123.75 L153.97 M 156.42 N 3.13 (s, 2H) 55.12 O 2.26 (s, 3H) 45.27 P 2.69 (t, J =5.7 Hz, 2H) 51.82 Q 3.47 (t, J = 5.7 Hz, 2H) 49.66 R 3.28 (s, 3H) 36.88

The NOESY data showed a weak NOE between the protons of the amidinemethyl group and the ortho-situated aromatic proton of the amide phenylgroup (labeled R and C, respectively, Diagram 2). Additionally, a strongNOE was observed between the methylene protons alpha to the amidinegroup and the ortho-positioned aromatic proton of the core (labeled Nand K, respectively, Diagram 2). Taken together, these data furthersupport the assignment of the E-stereochemistry of ML336.

Furthermore, it is expected that(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide(as well as those compounds of Formula III where R₃ is not hydrogen)will readily isomerize to the corresponding E isomer in conditionsfacilitating proton transfer, such as physiological conditions, proticsolvents, and aprotic polar solvents, including acid/base catalyzedconditions. Without being bound by theory, this expectation is foundednot only on the allylic strain of such systems, but also on the basicityof the amidine, the acidity of the amide, and the experimental resultsprovided herein.

Chemical Stability of ML336:

ML336 was evaluated for susceptibility to nucleophilic addition andformation of conjugates by treatment with dithiothreitol (DTT). FIG. 1represents the time course experiment with ML336 under variousconditions. ML336 was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) andindependently incubated at room temperature with no nucleophile presentor 50 μM dithiothreitol (DTT). The test reactions were sampled everyhour for eight hours and analyzed by LCMS. The analytical LCMS systemutilized for the analysis was a Waters Acquity system with UV-detectionand mass-detection (Waters LCT Premier). The analytical methodconditions included a Waters Acquity HSS T3 C18 column (2.1×50 mm, 1.8m) and elution with a linear gradient of 1% water to 100% CH₃CN at 0.6mL/min flow rate. Peaks on the 214 nm chromatographs were integratedusing the Waters OpenLynx software. Absolute areas under the curve werecompared at each time point to determine relative percent parentremaining. The masses of potential adducts and dimers of ML336 weresearched for in the final samples to determine if any detectable adductformed or dimerization had occurred. All samples were prepared induplicate. Ethacrynic acid, a known Michael acceptor, was used as apositive control. In the case of ML336, no adducts were detected at anytime point using LCMS detection.

Table 1 summarizes the percent remaining of ML336 at the endpoints ofeach run in each experiment.

TABLE 1 Percent ML336 Averaged Percent Remaining ML336 Remaining TestCondition Run after 8 h after 8 h ML336 without nucleophile 1 96.1393.92 (control) ML336 without nucleophile 2 91.70 (control) ML336 with5X DTT 1 100.36 98.96 ML336 with 5X DTT 2 97.55

Synthesis of(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide

(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide

(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamidewas synthesized according to a slight modification of the aboveprocedures. To a stirred solution of tert-butyl(2-(((3-(4-methoxyphenyl)-6-nitro-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (250 mg, 0.50 mmol) in CH₂Cl₂ (10 mL) was slowly addedTFA (0.39 mL, 5.02 mmol). The mixture was stirred at rt for 3 h, andthen the reaction was quenched with saturated solution of NaHCO₃. Theorganic phase was separated and the aqueous layer was extracted withCH₂Cl₂ (2×20 mL). The combined organic phase was dried with anydrousNa₂SO₄, concentrated, and purified by flash chromatography (CombiFlash,24 g silica, 0-5% MeOH/CH₂Cl₂) to give(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide(96 mg, 48%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.9 (s, 1H),9.14 (d, J=2.8 Hz, 1H), 8.13 (dd, J=8.8, 2.8 Hz, 1H), 7.54-7.52 (m, 2H),6.90-6.88 (m, 2H), 6.79 (d, J=8.8 Hz, 1H), 3.80 (s, 3H), 3.47 (t, J=5.4Hz, 2H), 3.26 (s, 3H), 3.13 (s, 2H), 2.69 (t, J=5.7 Hz, 2H), 2.26 (s,3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.52, 156.53, 156.42, 154.05, 142.93,131.53, 127.68, 126.41, 126.36, 123.84, 121.86, 121.86, 114.40, 77.55,77.23, 76.91, 55.66, 55.29, 52.00, 49.83, 45.43, 37.03. LCMS retentiontime: 3.169 min, purity at 254 nm=99.4%. HRMS m/z calculated forC₁₉H₂₂N₅O₃ [M⁺+H] 398.1828, found 388.1840.

Synthesis of(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide

Methyl 2-amino-5-cyanobenzoate.

Palladium (II) acetate (0.063 g, 0.28 mmol, 0.1 eq), DPPF (0.16 g, 0.28mmol, 0.1 eq), K₂CO₃ (1.16 g, 8.40 mmol, 3 eq) and Et₃N (0.39 mL, 2.80mmol, 1.0 eq) were added to a solution of 4-amino-3-iodobenzonitrile(0.68 g, 2.8 mmol) in MeCN (15 mL) and MeOH (7.5 mL). The reactionmixture was purged with N₂, the flask was capped, and a ballooncontaining CO was attached. After bubbling CO gas into the solutionthrough a needle attached to the balloon for 5 min, the mixture washeated under a CO balloon at 60° C. overnight. The mixture was dilutedwith EtOAc (200 mL) and filtered. The filtrate was washed with water(3×40 mL), brine (1×), dried (Na₂SO₄) and filtered. Solvent wasevaporated in vacuo. The residue was purified by flash chromatography onsilica gel to provide methyl 2-amino-5-cyanobenzoate (0.268 g, 54%) as awhite solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J=2.0 Hz, 1H),7.43 (dd, J=8.7, 2.1 Hz, 1H), 6.67 (d, J=8.6 Hz, 1H), 6.33 (s, 2H), 3.88(s, 3H).

2-amino-5-cyanobenzoic acid

To a solution of methyl 2-amino-5-cyanobenzoate (0.120 g, 0.68 mmol) inTHF (3.5 ml), was added a solution of lithium hydroxide (0.033 g, 1.36mol, 2.0 eq) in H₂O (3.5 ml). After stirring at rt for 3 h, the reactionmixture was concentrated. The residue was diluted with H₂O (5 ml) andacidified to pH-3 using 1M HCl. The precipitate was collected byfiltration, washed with H₂O and dried under air. 2-amino-5-cyanobenzoicacid (0.11 g, 95%) was obtained as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.04 (d, J=2.1 Hz, 1H), 7.70-7.33 (m, 3H), 6.86 (d, J=8.8 Hz,1H).

2-(chloromethyl)-4-oxo-4H-benzo[d] [1,3]oxazine-6-carbonitrile

To a solution of 2-amino-5-cyanobenzoic acid (0.10 g, 0.617 mmol, 1 eq)in CH₂Cl₂ (1.0 mL) was added triethylamine (0.3 mL, 2.15 mmol, 3.5 eq),and then the mixture was cooled to 0° C. in an ice bath, and a solutionof chloroacetyl chloride (0.16 mL, 2.0 mmol, 3.3 eq) in CH₂Cl₂ (1.5 mL)was slowly added. The reaction mixture was stirred at rt for 16 h. Thesolvent was removed in vacuo and water was added to the remaining solid.The solid was filtered, rinsed with water (10 mL) and dried under air toafford 2-(chloromethyl)-4-oxo-4H-benzo[d][1,3]oxazine-6-carbonitrile:(0.15 g, 99%) as a yellow solid. The product was used in the followingstep without further purification. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s,1H), 8.70 (d, J=8.8 Hz, 1H), 8.39 (d, J=2.1 Hz, 1H), 8.09 (dd, J=8.8,2.1 Hz, 1H), 4.53 (s, 2H).

2-(chloromethyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile

To a microwave vial containing a solution of2-(chloromethyl)-4-oxo-4H-benzo[d][1,3]oxazine-6-carbonitrile (0.14 g,0.59 mmol, 1 eq) in CH₂Cl₂ (0.8 mL) was added phosphorus oxychloride(0.11 mL, 1.17 mmol, 2 eq) and a solution of aniline (0.07 mL, 0.76mmol, 1.3 eq) in CH₂Cl₂ (0.8 mL). The resulting mixture was heated in aMW reactor at 150° C. for 15 min. The reaction mixture was slowlyquenched with saturated NaHCO₃(3 mL). The precipitate was filtered,rinsed with water (8 mL) and dried under air to afford2-(chloromethyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile(0.138 g, 80%) as a burnt-orange solid. ¹H NMR (400 MHz, Chloroform-d) δ8.63 (dd, J=2.0, 0.6 Hz, 1H), 8.03 (dd, J=8.5, 1.9 Hz, 1H), 7.89 (dd,J=8.5, 0.6 Hz, 1H), 7.69-7.58 (m, 3H), 7.41-7.35 (m, 2H), 4.30 (s, 2H).

(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide

To a solution of2-(chloromethyl)-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-carbonitrile(0.030 g, 0.10 mmol, 1 eq) in DMF (2 ml), was added K₂CO₃ (0.028 g, 0.20mmol, 2.0 eq) and N,N′-dimethylethanediamine (0.009 g, 0.10 mmol, 1.0eq). After stirring for 1.5 h, the reaction mixture was purified bychromatography to afford(E)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide(0.007 g, 20%) as a light yellow solid. Mp: 167-169° C. ¹H NMR (400 MHz,Chloroform-d) δ 10.95 (s, 1H), 8.61 (d, J=2.1 Hz, 1H), 7.68-7.62 (m,2H), 7.59 (dd, J=8.2, 2.1 Hz, 1H), 7.42-7.35 (m, 2H), 7.15 (tt, J=8.0,2.1 Hz, 1H), 6.81 (d, J=8.3 Hz, 1H), 3.47 (d, J=4.1 Hz, 2H), 3.29 (s,3H), 3.12 (s, 2H), 2.69 (d, J=4.1 Hz, 2H), 2.28 (s, 3H). ¹³C NMR (101MHz, CDCl₃) δ 162.86, 156.28, 152.07, 138.24, 136.09, 134.50, 129.10,126.64, 124.18, 124.09, 120.24, 118.95, 105.83, 55.07, 51.90, 49.65,45.32, 36.81. LCMS purity: 99.5%. LCMS retention time: 3.106 min. HRMSm/z calculated for C₂₀H₂₁N₅O [M⁺+H]: 348.1746, found 348.1820.

Synthesis of(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide

tert-Butyl ethyl(2-(methylamino)ethyl)carbamate

(2-aminoethyl)ethylcarbamic acid tert-butyl ester (Chem-Impex, 377 mg,2.00 mmol, 1 eq) was dissolved in toluene (5 mL), and then benzaldehyde(0.26 mL, 2.56 mmol, 1.3 eq) was added dropwise at rt. The reactionflask was equipped with a Dean-Stark trap and a reflux condenser. Themixture was heated at 150° C. for 1.5 hours. After cooling to rt, theDean-Stark trap was removed and a solution of methyl p-toluenesulfonate(0.31 mL, 2.05 mmol, 1.03 eq) in toluene (0.5 mL) was added dropwise.The mixture was heated to gentle reflux at 125° C. for 15 hours. Aftercooling to rt, water (2 mL) was added and the mixture was heated at 80°C. for 30 min. After cooling to rt, the biphasic layers were separatedand 2 M aq. KOH (4 mL) was added to the aq. layer. The product wasextracted from the aq. layer with CH₂Cl₂ (3×15 mL) and dried with Na₂SO₄to give tert-butyl ethyl(2-(methylamino)ethyl)carbamate (171 mg, 42%) asa clear, pale yellow oil, which was used in the next step withoutfurther purification.

tert-Butylethyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate

Using a MW vial, 2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one(177 mg, 0.56 mmol, 1 eq) was dissolved in MeCN (2.6 mL). Potassiumcarbonate (157 mg, 1.14 mmol, 2 eq), tert-butylethyl(2-(methylamino)ethyl)carbamate (161 mg, 0.80 mmol, 1.4 eq), andpotassium iodide (36 mg, 0.22 mmol, 0.4 eq) were added successively tothe reaction vial. The mixture was heated in a MW reactor at 80° C. for10 min. The product was purified by flash chromatography (CombiFlash, 12g silica, 0-10% MeOH/CH₂Cl₂) to give tert-butylethyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate(88 mg, 33%) as a clear, dark red oil. ¹H NMR (400 MHz, CDCl₃) δ 9.07(d, J=2.7 Hz, 1H), 8.53 (dd, J=8.9, 2.6 Hz, 1H), 7.88 (d, J=8.9 Hz, 1H),7.59-7.52 (m, 3H), 7.33-7.29 (m, 2H), 3.37 (s, 2H), 3.20-3.00 (m, 4H),2.52-2.42 (m, 2H), 2.22 (s, 3H), 1.40 (br s, 9H), 1.02 (t, J=7.2 Hz,3H).

(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide

To a solution of tert-butylethyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate(88 mg, 0.183 mmol) in CH₂Cl₂ (2.8 mL) was added TFA (1.2 mL, 15.67mmol). The reaction was stirred at rt for 45 min. After the reaction wascomplete, water (7 mL) and CH₂Cl₂ (7 mL) were added and the reactionmixture was adjusted to pH 10 using saturated aq. Na₂CO₃ (5 mL). Theproduct was extracted with CH₂Cl₂ (3×15 mL) and purified by flashchromatography (CombiFlash, 0-5% MeOH/CH₂Cl₂) to give(E)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide(18 mg, 23%) as a pale, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.66(s, 1H), 9.14 (d, J=2.8 Hz, 1H), 8.16 (dd, J=8.8, 2.8 Hz, 1H), 7.65-7.56(m, 2H), 7.38-7.33 (m, 2H), 7.16-7.10 (m, 1H), 6.81 (d, J=8.8 Hz, 1H),3.78 (q, J=7.1 Hz, 2H), 3.46 (t, J=5.6 Hz, 2H), 3.12 (s, 2H), 2.68 (t,J=5.6 Hz, 2H), 2.25 (s, 3H), 1.30 (t, J=7.2 Hz, 3H). ¹³C NMR (101 MHz,CDCl₃) δ 162.92, 155.87, 154.58, 142.87, 138.20, 129.17, 127.84, 126.57,126.08, 124.47, 123.98, 120.68, 55.39, 51.99, 46.78, 45.33, 43.56,12.16. LCMS retention time: 3.344 min, purity at 214 nm=88%. HRMS m/zcalculated for C₂₀H₂₄N₅O₃ [M⁺+H] 382.1879, found 382.1915. Pale yellowsolid, mp 150-157° C. (decomposition).

Synthesis of6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one

tert-Butyl4-((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)piperazine-1-carboxylate

2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (253 mg, 0.801mmol, 1 eq) was dissolved in CH₂Cl₂(3.6 mL). Potassium carbonate (224mg, 1.621 mmol, 2 eq), 1-piperazinecarboxylic acid, 1,1-dimethylethylester (224 mg, 1.203 mmol, 1.5 eq), and potassium iodide (50 mg, 0.301mmol, 0.4 eq) were added successively to the reaction vial. The mixturewas heated in a MW reactor at 80° C. for 5 min. The product was purifiedby flash chromatography (CombiFlash, 0-50% EtOAc/hexanes) to givetert-butyl4-((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)piperazine-1-carboxylate(103 mg, 28%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J=2.5Hz, 1H), 8.56 (dd, J=9.0, 2.7 Hz, 1H), 7.87 (d, J=8.9 Hz, 1H), 7.60-7.51(m, 3H), 7.35-7.29 (m, 2H), 3.33-3.27 (m, 6H), 2.27 (t, J=4.8 Hz, 4H),1.43 (s, 9H).

6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one

tert-butyl4-((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)piperazine-1-carboxylate(73 mg, 0.157 mmol) was dissolved in CH₂Cl₂(2.4 mL) and then TFA (1 mL,13.06 mmol) was added dropwise. The mixture was stirred at rt for 2 h.After the reaction was complete, water (6 mL) and CH₂Cl₂(6 mL) wereadded, and the reaction mixture was adjusted to pH 10 using saturatedaq. Na₂CO₃ (4 mL). The product was extracted with CH₂Cl₂(3×12 mL) andpurified by flash chromatography (CombiFlash, 0-10% MeOH/DCM) to give6-nitro-3-phenyl-2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one (49 mg,85%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J=2.5 Hz,1H), 8.61 (dd, J=9.0, 2.7 Hz, 1H), 7.93 (d, J=9.0 Hz, 1H), 7.58-7.49 (m,5H), 3.25 (s, 2H), 2.66 (t, J=4.5 Hz, 4H), 2.19 (t, J=4.7 Hz, 4H). LCMSretention time: 2.607 min, purity at 214 nm=99.2%. HRMS m/z calculatedfor C₁₉H₂₀N₅O₃ [M⁺+H] 366.1561, found 366.1567. White solid, mp 190-195°C.

Synthesis of(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide

2-(chloromethyl)-3-(3-fluorophenyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile

To a microwave vial containing2-(chloromethyl)-4-oxo-4H-benzo[d][1,3]oxazine-6-carbonitrile (0.147 g,0.616 mmol, 1 eq) was added CH₂Cl₂ (0.7 mL). Phosphorus oxychloride(0.12 mL, 1.23 mmol, 2.0 eq) and a solution of 3-fluoroaniline (0.08 mL,0.80 mmol, 1.3 eq) in CH₂Cl₂ (1.0 mL) was added and the mixture washeated in a MW reactor at 150° C. for 15 min. The reaction mixture wasslowly quenched with saturated NaHCO₃(5 mL). The precipitate wasfiltered, rinsed with water (8 mL) and dried under air to afford2-(chloromethyl)-3-(3-fluorophenyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile(0.150 g, 78%) as a burnt-orange solid. ¹H NMR (400 MHz, Chloroform-d) δ8.60 (d, J=2.0 Hz, 1H), 8.01 (dd, J=8.5, 2.0 Hz, 1H), 7.87 (d, J=8.5 Hz,1H), 7.59 (td, J=8.2, 5.9 Hz, 1H), 7.32 (tdd, J=8.3, 2.5, 1.0 Hz, 1H),7.17 (ddd, J=8.0, 2.1, 1.0 Hz, 1H), 7.13 (dt, J=8.6, 2.3 Hz, 1H), 4.29(s, 2H).

(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide

To a solution of2-(chloromethyl)-3-(3-fluorophenyl)-4-oxo-3,4-dihydroquinazoline-6-carbonitrile(0.020 g, 0.064 mmol, 1.0 eq) in DMF (0.64 ml), was added K₂CO₃ (0.018g, 0.128 mmol, 2.0 eq) and N,N′-dimethylethanediamine (0.007 g, 0.083mmol, 1.3 eq). After stirring for 2 h, the reaction mixture was purifiedby chromatography to afford(E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide(0.010 g, 43%) as a light yellow solid. Mp: 179-182° C. ¹H NMR (400 MHz,Chloroform-d) δ 11.09 (s, 1H), 8.59 (d, J=2.1 Hz, 1H), 7.63-7.55 (m,2H), 7.35-7.24 (m, 2H), 6.89-6.79 (m, 2H), 3.48 (t, J=4.1 Hz, 2H), 3.29(s, 3H), 3.13 (s, 2H), 2.71 (t, J=4.1 Hz, 2H), 2.29 (s, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 164.30, 163.01, 161.87, 156.49, 152.01, 139.84,139.73, 136.11, 134.67, 130.13, 130.04, 126.29, 124.13, 118.84, 115.39,110.95, 110.74, 107.82, 107.56, 55.12, 51.89, 49.68, 45.33, 36.84.Purity: 99.3%. LCMS retention time: 3.210 min. HRMS m/z calculated forC₂₀H₂₀FN₅O [M⁺+H]: 366.1653, found 366.1727.

Assay Conditions

HTS Primary and Confirmatory Assay Using TC-83.

Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were culturedand maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS(Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintainedat 37° C., 5.0% CO₂ to 100% confluence being passaged 1:4 every 3-4days. For cell plating, cells were detached from flask bottom by usingTrypsin-EDTA solution and then re-suspended in a growth media. Cellswere passaged no more than ten times after being thawed.

VEEV culture: VEEV TC-83 was used for screening. The VEEV stock wasprepared in Vero76 cells using an initial stock obtained from Dr. Chung.

Compound Dosing/Plating: The positive control was MPA at 10 uM finalwell concentration. The compounds were diluted in complete growth mediumto 6× concentrated dosing solution which was dispensed into 384-wellblack clear-bottom tissue culture treated plates (5 μL volume).

Single Dose Compound Preparation: The MLSMR library was plated at 25 μMsingle dose concentration.

Dose Response Compound Preparation: The compounds were tested in a doseresponse format using a 1:2 serial dilution with the highestconcentrations starting at 25 M and extending to 0.05 μM over a 10-plate1:2 serial dilution pattern. DMSO and compounds were diluted in assaymedia to 6× and 5 μL was dispensed to assay plates. The final DMSO inthe assay for all screening concentrations was 0.25%.

Virus Addition: VEEV stock was diluted in the culture media to 6.44pfu/ml. (MOI 4e-5)

VEEV and Cell Plating: 3,000 cells/well alone or with VEEV virus at thepreviously indicated dilution (180,000 cells/ml) were plated in 25 uLusing a Matrix WellMate. All additions were done using a Matrix WellMatehoused in a class II Biosafety Cabinet within the BSL-2 laboratory. Theplates were incubated in an actively humidified incubator with 5.0% CO₂at 37° C. for 72 h and 95% humidity.

Endpoint Read: The assay plates were equilibrated to room temperaturefor 30 minutes and then an equal volume of CellTiter-Glo reagent(Promega Inc.) was added to each well. Plates were incubated for 10 minat room temperature and luminescence was measured using a Perkin ElmerEnvision multi-label reader.

Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83.

Cell Culture: Vero 76 (CRL-1587, ATCC) were purchased from ATCC andmaintained in 37° C. incubator with 5% CO₂. The cells were cultured in acomplete media (Minimum Essential Media with Earle's salt and 10% fetalbovine serum). Cells were passaged once a week and harvested from flasksusing 0.05% trypsin-EDTA.

Assay Media—Preparation of Complete DMEM media: 5 mL Pen/Strep (Gibco,Cat. No. 15149) and 50 mL of heat-inactivated FBS (Gibco, Cat. No.10082147) was added to 500 mL of Dulbecco's Modified Eagle Medium(Gibco, Cat. No. 11995-073).

Virus: TC-83 strain was obtained from Dr. Brett Beitzel from UnitedState Army Research Medical Research Institute for Infectious diseasesand amplified in BHK C-21 cell line once.

Dose Response Compound Preparation: For dose response screening,compounds or carrier control (DMSO) were diluted to 3× in Complete DMEMmedia. Test compounds were serially diluted 1:2 resulting in an 8 pointdose response dilution series. (final plate well concentration rangingfrom 50 uM to 0.39 μM and a final DMSO concentration of 0.25%). 30 l ofeach dilution was dispensed to assay plates (0.75% DMSO) in duplicate.

Control Drug: The positive control drug for this assay, mycophenolicacid was solubilized in DMSO. It was diluted and added to the assayplates as described for test compounds. Final concentration forribavirin was 10 M. All wells contained 0.25% DMSO.

Assay Set up: Vero 76 cells were plated in 96-well plates at a densityof 15,000 cells per well in a volume of 45 μL of DMEM complete media.The cells were grown for 24 hours prior to testing in a 5% CO₂, 37 Ccell culture incubator. Viruses, TC-83 strain, was diluted in cellculture medium to be 750 pfu/15 uL (0.05 MOI) and then added to theplates at a volume of 15 μL per well. The plates were incubated for 48hours in a 37° C. incubator with 5% CO₂.

Endpoint Read: Following the two day incubation period, the assay plateswere equilibrated to room temperature for 10 min and an equal volume (90μL) of Cell Titer-Glo reagent (Promega Inc.) was added to each wellusing a Microflow (Biotek, VT) and plates were incubated for anadditional 10 min at room temperature. At the end of the incubation,luminescence was measured using a Synergy4 Multimode plate reader(Biotek, VT) with an integration time of 0.2 s.

Data Analysis: Results are reported as percent (%) CPE inhibition andwere calculated using the following formula: % CPE inhibition=100*(TestCmpd−Med Virus)/(Med Cells−Med Virus). Four ribavirin positive controlwells were included on each plate for quality control purposes. Toquantify the viral cytopathic effect, IC50s were calculated for eachsubstance using the 4 parameter Levenburg-Marquardt algorithm with theminimum and maximum parameters locked at 0 and 100, respectively.

Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, V3526.

Cell Culture: Vero 76 (CRL-1587, ATCC) were purchased from ATCC andmaintained in 37° C. incubator with 5% CO₂. The cells were cultured in acomplete media (Minimum Essential Media with Earle's salt and 10% fetalbovine serum). Cells were passaged once a week and harvested from flasksusing 0.05% trypsin-EDTA.

Assay Media—Preparation of Complete DMEM media: 5 mL Pen/Strep (Gibco,Cat. No. 15149) and 50 mL of heat-inactivated FBS (Gibco, Cat. No.10082147) was added to 500 mL of Dulbecco's Modified Eagle Medium(Gibco, Cat. No. 11995-073).

Virus: V3526 strain. Virus was rescued from BHK C-21 cells that weretransfected with infectious V3526 RNA. The recued virus was amplified inBHK C-21 cells once and then used as a stock virus.

Dose Response Compound Preparation: For dose response screening,compounds or carrier control (DMSO) were diluted to 3× in Complete DMEMmedia. Test compounds were serially diluted 1:2 resulting in an 8 pointdose response dilution series. (final plate well concentration rangingfrom 50 μM to 0.39 μM and a final DMSO concentration of 0.25%). 30 L ofeach dilution was dispensed to assay plates (0.75% DMSO) in duplicate.

Control Drug: The positive control drug for this assay, mycophenolicacid was solubilized in DMSO. It was diluted and added to the assayplates as described for test compounds. Final concentration forribavirin was 10 M. All wells contained 0.25% DMSO.

Assay Set up: Vero 76 cells were plated in 96-well plates at a densityof 15,000 cells per well in a volume of 45 μL of DMEM complete media.The cells were grown for 24 hours prior to testing in a 5% CO₂, 37° C.cell culture incubator. V3526 VEEV virus, was diluted in cell culturemedium to be 750 pfu/15 uL (0.05 MOI) and then added to the plates at avolume of 15 μL per well. The plates were incubated for 48 hours in a37° C. incubator with 5% CO₂.

Endpoint Read: Following the two day incubation period, the assay plateswere equilibrated to room temperature for 10 min and an equal volume (90μL) of Cell Titer-Glo reagent (Promega Inc.) was added to each wellusing a Microflow (Biotek, VT) and plates were incubated for anadditional 10 min at room temperature. At the end of the incubation,luminescence was measured using a Synergy4 Multimode plate reader(Biotek, VT) with an integration time of 0.2 s.

Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 andother Alphaviruses (Trinidad Donkey).

Biosafety and Biosecurity: All experiments with VEEV Trinidad donkey(TrD) strain was done in the Regional Biocontainment Laboratory (RBL) inUniversity of Louisville. All procedures were done in compliance withSelect Agent Rules.

Cell Culture: Vero 76 cells obtained from ATCC (CRL-1586) were culturedand maintained in in Dulbecco's Modified Eagle's Medium (DMEM) with 4500mg/L glucose, 2 mM L-glutamine, and 10% FBS (culture media). The cellsare maintained at 37° C., 5.0% CO₂ to 100% confluence being passagedevery three to seven days. For cell plating, cells were detached fromflask bottom by using 0.05% Trypsin-EDTA solution and then re-suspendedin a growth media.

VEEV culture: VEEV TrD strain was used for screening. The VEEV TrD stockwas prepared in Vero 76 cells using an initial stock obtained from WorldReference Center for Emerging Viruses and Arboviruses (Dr. Robert Tesh).Briefly, cells were grown in two T-175 flasks to 50% confluence in aculture media. The cells were infected with 1 mL of diluted virus stock(1:10 dilution of the original stock) per T175 for 1.5 hours and thenwashed, and replenished with 25 mL media. The cells were incubated for 2days in an incubator at 37° C., 5% CO₂ and high humidity. Thesupernatant was harvested and the cell debris pelleted by centrifugingat 1,000 rpm for 5 minutes at 18° C. The supernatant was aliquoted (1 mLper tube) and stored at −80° C. These virus stocks were titrated in Vero76 cells using an agarose overlay plaque method and the titers were2.0×E10 pfu/mL.

Dose Response Compound Preparation: The compounds were tested in a doseresponse format using a 1:2 serial dilution with the highestconcentrations starting at 12.5 μM and extending to 0.09 μM over 8points 1:2 serial dilution pattern. DMSO and compounds were diluted inassay media to 3× and 30 μL was dispensed to assay plates (see below).The final DMSO in the assay for all screening concentrations was 0.25%.

Assay Set up: 45 μl of Vero 76 cell suspension (267,000 cells/mL) wereplated in clear bottom black well 96-well plates and the plates wereincubated in an incubator at 37° C. in a humidified 5% CO₂ atmosphere.The next day, thirty uL of drugging media (0.75% DMSO) were added to theeach wells and the plates were incubated at 37° C. for an hour. Theplates were transferred into a BSL-3 lab in the RBL. Each well receivedfifteen L of VEEV TrD virus diluted in Complete media (40,000 pfu/mL,final 0.05 MOI). For the cell control wells, Complete media were addedinstead of virus solution. Drug plating was conducted using a EVO100,96MAC (Tecan) and virus solution was added using MicroFlo (Biotek). Theassay plates were incubated for two days at 37° C., 5% CO₂ and 90%relative humidity.

Endpoint Read: The assay plates were equilibrated to room temperaturefor 30 minutes and then an equal volume of CellTiter-Glo reagent(Promega Inc.) was added to each well. Plates were incubated for 10 minat room temperature and luminescence was measured using a Synergy4 HTmulti-label reader.

Vero76 Cytotoxicity Assay for VEEV Compounds

Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were culturedand maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS(Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintainedat 37° C., 5.0% CO₂ to 100% confluence being passaged 1:4 every 3-4days. For cell plating, cells were detached from flask bottom by usingTrypsin-EDTA solution and then re-suspended in a growth media. Cellswere passaged no more than ten times after being thawed.

Compound Dosing/Plating: Carrier control/compounds were diluted incomplete growth medium to prepare a 6× concentrated dosing solutionwhich was dispensed into 384-well black clear-bottom tissue culturetreated plates (5 μL volume).

Cell Plating: Twenty-five uL of complete growth medium containing 3000cells were dispensed per well. Plates were incubated at 37° C., 5% CO₂for 72 h prior to endpoint detection.

Endpoint/Detection: At the end of the treatment period, assay plateswere removed from the incubator and equilibrated to room temperature for10 min. Thirty uL of Cell Titer Glo reagent was added and plates wereincubated for an additional 10 min in the dark. At the end of theincubation, assay plates were analyzed using a PerkinElmer Envisionmicroplate reader in luminescence mode with an integration time of 0.1s.

Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV(Strain Trinidad Donkey)

Biosafety and Biosecurity: All experiments with VEEV Trinidad donkeystrain was done in the Regional Biocontainment Laboratory in Universityof Louisville. All procedures were done in compliance with Select AgentRules.

Cell Culture: Vero 76 cells (ATCC; CRL1586) were cultured in Dulbecco'sModified Eagle's Medium (DMEM) with 4500 mg/L glucose, 2 mM L-glutamine,and 10% FBS (culture media). The cells are maintained at 37° C., 5.0%CO₂ to 100% confluence being passaged every three to seven days. Forcell plating, cells were detached from flask bottom by using 0.05%Trypsin-EDTA solution and then re-suspended in a growth media.

VEEV culture: Trinidad Donkey (TrD) was used for screening. The VEEV TrDstock was prepared in Vero 76 cells using an initial stock obtained fromWorld Reference Center for Emerging Viruses and Arboviruses (Dr. RobertTesh). Briefly, cells were grown in two T-175 flasks to 50% confluencein a culture media. The cells were infected with 1 mL of diluted virusstock (1:10 dilution of the original stock) per T175 for 1.5 hours andthen washed, and replenished with 25 mL media. The cells were incubatedfor 2 days in an incubator at 37° C., 5% CO₂ and high humidity. Thesupernatant was harvested and the cell debris pelleted by centrifugingat 1,000 rpm for 5 minutes at 18° C. The supernatant was aliquoted (1 mlper tube) and stored at −80° C. These virus stocks were titrated in Vero76 cells using an agarose overlay plaque method and the titer was2.0×E10 pfu/ml.

Cell Plating: Vero 76 cells were seeded at 70% confluence in a 12-wellplate in a volume of lmL and incubated for overnight at 37° C. with 5%CO₂ and high humidity.

Virus Addition: The cells were infected with virus by adsorption for anhour. Cell culture media in the 12-well plates were removed completelyand the cells were infected with either mock virus (media only) or VEEV.VEEV stock was diluted in the culture media to 9×E03 pfu/ml and 200 μLwas added to the test wells and the virus control wells (final MOI of0.1). The plates were incubated in an actively humidified incubator with5.0% CO₂ at 37° C. for one hour. During the incubation, plates weregently rocked every 20 min. to ensure the coverage of the cells withvirus. After the adsorption, the cells were rinsed lmL of PBS per welland then replenished with the media containing testing articles. Alladditions were performed in a class II Biosafety Cabinet. The plateswere incubated in an actively humidified incubator with 5.0% CO₂ at 37°C. for 48 h.

Control and Drug Preparation: Carrier Control consisted of DMSO dilutedin assay media to 0.25% and 1000 μL was dispensed to both cell and viruscontrol wells of 12-well tissue culture treated plates. Test compoundswere diluted in media to be at target concentration with a DMSOconcentration of 0.25%.

Titration of Progeny viruses (Mini plaque assay): Titer of progenyviruses produced from the cell was measured by a mini plaque assay in96-well plate format. Fresh Vero 76 cells were seeded and grown in96-well plates overnight. The cell culture supernatants from the 96-wellplates were emptied and the cells were infected with 25 μL of 10-foldserial dilutions of progeny virus containing medium from respectivesamples (drug treated or untreated). The plates were incubated for onehour in an incubator at 37° C., 5% CO₂. The cells were rinsed with 100μL per well of PBS once and then replenished with DMEM with 0.75%methyl-cellulose and 10% FBS. The cell plates were incubated at 37° C.,5% CO₂, and high humidity for an additional three days. Crystal violetsolution with 4% paraformaldehyde was used to developed plaques in thewells. The assay plates were equilibrated to room temperature for 10minutes and then an equal volume of the crystal violet solution wasadded to each well. The plates were incubated for 60 min at roomtemperature and stained one more time. After a wash the plates withwater, the number of plaques in each wells were determined by a visualcounting. Virus titers were calculated by: No. of plaques×10E (dilutionfold at the counting)*1000/25 (pfu/mL). Compounds treatment were done ina duplicate independently and mean from duplicates of titration wasused. Log reduction of titer was calculated by: Log₁₀(titer of Poscontrol)−Log₁₀(titer of sample).

Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitorsin a Cell-Based Anti-Respiratory Synctial Virus (RSV) Assay

Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type)were maintained as adherent cell lines in DMEM with 2 mM L-glutamine and10% fetal bovine serum (FBS) at 37° C. in a humidified 5% CO₂atmosphere. Cells were passaged as needed and harvested from flasksusing 0.05% trypsin-EDTA.

Assay Media—Preparation of Complete DMEM/F12: DMEM/F12 (Invitrogen, Cat.No. 11320) was supplemented with 5 mL of Pen/Strep (Invitrogen, Cat. No.10378016), 5 mL of 200 mM glutamine (Invitrogen, Cat No. 25030-081), and10 mL of HI-FBS was added per 500 mL of media.

RSV culture: Human respiratory syncytial virus (HRSV) strain Long (ATCCVR-26) was used for screening. The RSV stock was prepared in HEp-2 cellsusing an initial stock obtained from ATCC. Briefly, HEp-2 cells weregrown in two T-175 flasks to 50% confluence in Dulbecco's Modified EagleMedium: Nutrient Mixture F-12 (DMEM/F12), pH 7.5 with 2.5 mML-glutamine, 2% FBS and 125 U of penicillin, 125 ug of streptomycin perml. 0.2 mL of RSV was added to 25 ml of CDMEM/F12. After three daysincubation at 37° C., 5% CO₂ and high humidity, the supernatant washarvested and the cell debris pelleted by centrifuging at 1,000 rpm for5 minutes at 18° C. Trehalose and FBS were added to a finalconcentration of 10% each and the supernatant was aliquoted (1 ml pertube) and stored at −80° C. These virus stocks were titrated in HEp-2cells using an agarose overlay plaque method and the titer was 1.0 E7pfu/ml.

Dose Response Compound Preparation: For dose response screening,compounds or carrier control (DMSO) were diluted to 3× in CompleteDMEM/F12. Test compounds were serially diluted 1:2 resulting in an 8point dose response dilution series. (final plate well concentrationranging from 50 μM to 0.39 μM and a final DMSO concentration of 0.25%).39 L of each dilution was dispensed to assay plates (0.75% DMSO) intriplicate.

Control Drug: The positive control drug for this assay, MPA wassolubilized in DMSO.

Preparation of HEp-2 cells: Cells were harvested and resuspended to267,000 cells per ml in Complete DMEM/F12.

Assay Set up: Forty five ul of HEp-2 cell suspension (12,000 cells/well)were plated in clear bottom black well 96-well plates and the plateswere incubated in an incubator at 37° C. in a humidified 5% CO₂atmosphere. The next day, 30 μL of drugging media (0.75% DMSO) wereadded to the each wells and the plates were incubated at 37° C. for anhour. Each well received 15 μL of RSV diluted in Complete DMEM/F12 media(40,000 pfu/mL, final 0.05 MOI). For the cell control wells, CompleteDMEM/F12 media were added instead of virus solution. Drug plating wasconducted using a EVO100, 96MAC (Tecan) and virus was added usingMicroFlo (Biotek). The assay plates were incubated for five days at 37°C., 5% CO₂ and 90% relative humidity.

Endpoint Read: The assay plates were equilibrated to room temperaturefor 30 minutes and then an equal volume of CellTiter-Glo reagent(Promega Inc.) was added to each well. Plates were incubated for 10 minat room temperature and luminescence was measured using a Synergy4multi-label reader.

HEp2 Cytotoxicity Assay for VEEV Compounds

Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type)were maintained as adherent cell lines in DMEM with 2 mM L-glutamine and10% fetal bovine serum (FBS) at 37° C. in a humidified 5% CO₂atmosphere. Cells were passaged as needed and harvested from flasksusing 0.05% trypsin-EDTA.

Assay Media—Preparation of Complete DMEM/F12: DMEM/F12 (Invitrogen, Cat.No. 11320) was supplemented with 5 mL of Pen/Strep (Invitrogen, Cat. No.10378016), 5 mL of 200 mM glutamine (Invitrogen, Cat No. 25030-081), and10 mL of HI-FBS was added per 500 mL of media.

Dose Response Compound Preparation: For dose response screening,compounds or carrier control (DMSO) were diluted to 3× in CompleteDMEM/F12. Test compounds were serially diluted 1:2 resulting in an 8point dose response dilution series. (final plate well concentrationranging from 50 μM to 0.39 μM and a final DMSO concentration of 0.25%).30 L of each dilution was dispensed to assay plates (0.75% DMSO) intriplicate.

Control Drug: The positive control drug for this assay, MPA wassolubilized in DMSO.

Preparation of HEp-2 cells: Cells were harvested and resuspended to267,000 cells per ml in Complete DMEM/F12.

Assay Set up: Forty five ul of HEp-2 cell suspension (12,000 cells/well)were plated in clear bottom black well 96-well plates and the plateswere incubated in an incubator at 37° C. in a humidified 5% CO₂atmosphere. The next day, 30 μL of drugging media (0.75% DMSO) wereadded to the each wells and the plates were incubated at 37° C. for anhour. Each well then received 15 μL of Complete DMEM/F12 media. For thecell control wells, Complete DMEM/F12 media were added instead of virussolution. Drug plating was conducted using a EVO100, 96MAC (Tecan) andvirus was added using MicroFlo (Biotek). The assay plates were incubatedfor five days at 37° C., 5% CO₂ and 90% relative humidity.

Endpoint Read: The assay plates were equilibrated to room temperaturefor 30 minutes and then an equal volume of CellTiter-Glo reagent(Promega Inc.) was added to each well. Plates were incubated for 10 minat room temperature and luminescence was measured using a Synergy4multi-label reader.

Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 andother Alphaviruses (Chikungunya virus).

Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were culturedand maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS(Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintainedat 37° C., 5.0% CO₂ to 100% confluence being passaged 1:4 every 3-4days. For cell plating, cells were detached from flask bottom by usingTrypsin-EDTA solution and then re-suspended in a growth media. Cellswere passaged no more than ten times after being thawed.

Compound Dosing/Plating: No positive control. The compounds were dilutedin complete growth medium to 6× concentrated dosing solution which wasdispensed into 96-well black clear-bottom tissue culture treated plates(25 μL volume).

Dose Response Compound Preparation: The compounds were tested in a doseresponse format using a 1:2 serial dilution with the highestconcentrations starting at 100 M and extending to 0.78 μM over a 8-dose1:2 serial dilution pattern. DMSO and compounds were diluted in assaymedia to 4× and 25 μL was dispensed to assay plates. The final DMSO inthe assay for all screening concentrations was 0.25%.

Virus Addition: CHIKV stock was diluted in the culture media to 100TCID50s/25 μL, and 25 μL were added to each test well.

Cell Plating and virus addition: 6,000 cells/well (120,000 cells/ml)were plated in 50 uL using a Matrix WellMate. All additions were doneusing a Matrix WellMate housed in a class II Biosafety Cabinet withinthe BSL-2 laboratory. The plates were incubated overnight in an activelyhumidified incubator with 5.0% CO₂ at 37° C. for 18 h and 95% humidity.Compounds (25 μL) were added after the cells had adhered to the plate,and CHIKV virus was added immediately after compound addition. Theplates were incubated for 72 h in an actively humidified incubator with5.0% CO₂ at 37° C. for 72 h and 95% humidity, and then endpoint reagentwas added.

Endpoint Read: The assay plates were equilibrated to room temperaturefor 30 minutes and then 100 μL of CellTiter-Glo reagent (Promega Inc.)was added to each well. Plates were incubated for 10 min at roomtemperature and luminescence was measured using a Perkin Elmer Envisionmulti-label reader.

Tested Compounds and Results

Testing of Known Compounds for VEEV Inhibitory Activity

A number of compounds were from patent and literature sources withreport VEEV inhibitory activity (Scheme 2); however, closer inspectionof the data and/or testing in the above-described CPE assay as well astheir dependency on host mediated mechanisms of action revealed that thecompounds of Scheme 2 suffer from a variety of disadvantages (Table 2).

TABLE 2 Assay provided Reported VEEV PubChem VEEV CPE CPE compound nameCID inhibition inhibition noted liability 1 ribavirin 37542 unknown CPE= not specific to viral 126 μM target 2 carbodine 459903 unknown NT notspecific to viral target 3 diclofenac 5018304 unknown >50 μM notspecific to viral sodium target; NSAID, off target effects 4 VX-497153241 CPE = NT not specific to viral 19.2 μM target 5 quinazolinone13182904 CPE = CPE > 25 μM mechanism 16.7 μM unknown 6 thienylpyrrole3240671 CPE < 100 μM CPE > 25 μM mechanism unknown 7 antioxidant 8066662 unknown CPE > 50 μM CYP450 (2C9, 2C19) inhibitor ~3 μM 8 didemnin44287859 unknown NT cyclic peptide (non-small molecule)

In all cases, the reported compounds are either weakly inhibitoryagainst VEEV (>25 μM) and/or do not act selectively on viral components.The interaction of these compounds on cellular components of the hostcan lead to undesirable toxicity. Ribavirin (Table 2, entry 1) andcarbodine (entry 2) are nucleoside anti-metabolite prodrugs with notableclinical toxicities and are not specific in their mechanism of action toviral targets. For example, ribavirin has a broad in vitro inhibitoryactivity against RNA viruses. The activities include 1) depleting thecellular GTP pool (IMPDH inhibitor), 2) increasing mutations in theviral genome, and 3) inhibiting the GTP capping enzyme. Direct,antiviral activity for ribavirin against Sindbis virus (anotheralphavirus) has been disclosed, but inhibitory activity for VEEV has notyet been reported. Our testing of ribavirin against VEEV in a CPE assayshowed an IC₅₀ of 126 μM. Diclofenac (Table 2, entry 3) is anonsteroidal anti-inflammatory drug (NSAID), thereby acting on cellularcomponents of the host, and was shown to not significantly inhibit VEEVin the CPE assay (>50 μM). Urea VX-497 (Table 2, entry 4) is a potent,reversible uncompetitive IMP dehydrogenase (IMPDH) inhibitor with modestVEEV activity. IMPDH catalyzes an essential step in the de novobiosynthesis of guanine nucleotides, and as such, VX-497 is known totarget cellular processes.

The quinazolinone structure (Table 2, entry 5) had a reported VEEV CPEIC₅₀ of 16.7 μM. Upon synthesis of the compound for assessment in ourinternal CPE assay; however, we found the IC₅₀ for this compound tobe >25 μM. SAR studies provided herein support that quinazolinone shouldnot be active for VEEV.

Thienylpyrrole (Table 2, entry 6) is reported to inhibit WEEV viralreplication with an IC₅₀=9.3 μM. VEEV inhibition is referred to (<100μM) for the compound class but is not explicitly reported for any onecompound. To dispel ambiguity, the compound was synthesized and assessedin our internal CPE assay, corroborating the finding with a VEEV IC₅₀>25μM.

Antioxidant 80 (Table 2, entry 7) is a broad spectrum antiviral agentdue to its antioxidant activity, and it has notable CYP450 (2C9, 2C19)inhibition to the tune of about 3 μM. This compound was purchased andpurified prior to submitting to the CPE assay, which resulted in a VEEVIC₅₀ of >50 μM.

Lastly, didemnin (Table 2, entry 8) was reported as an active compoundfor a VEEV screen; however, it is a large cyclic peptide and not aninhibitor of the present technology.

Compounds of Present Technology

Given the modest VEEV inhibition exhibited by the compounds in Table 2,their dependence on host targets, and unclear mechanisms of action, thecompounds of the present technology stand out as a first-in-class,potent small molecule VEEV inhibitors. In some embodiments, a potentinhibitor is an inhibitor with a potency <25 μM. In some embodiments,the potentcy is less than 16 μM. In some embodiments, the potentcy is alow nanomolar potency. In some embodiments, the compounds exhibit aselectivity index >1500 with respect to cytotoxicity, an ability toreduce viral titer, and a favorable in vitro pharmacokinetic profilewhich includes moderate blood-brain barrier (BBB) permeability. Withoutbeing bound by theory, the compounds of the present technology appear toact through a post-entry, viral-specific, mechanism of action byinhibiting viral replication through the nsP2 helicase. Thus, there arefewer off-target effects because the compounds target the virus and nothost.

The following compounds of the present technology were synthesizedaccording the above procedures or slight modifications thereof and werecharacterized by mass spectroscopy. Each compound gave the expected ¹Hand ¹³C NMR spectra, and gave the expected (M+H)⁺ peaks in the massspectrum.

Surprisingly, it was discovered that inclusion of an electronwithdrawing group at the C6 position of the molecule conferredsignificant CPE potency. Migration of the nitro group from the C6position to the C5-, C7- or C8-positions afforded analogs withoutsignificant CPE potency (>25 μM, Table 3).

TABLE 3

VEEV Cytotoxicity Assay mean (μM) n = # replicates) VEEV titer reductionIC₅₀ CC₅₀ Selectivity mean (log Entry R₃ n μM n μM (CC₅₀/IC₅₀) n log 16-NO₂ 33 0.78 7 >50 >63.3 2 6.36 2 7-NO₂ 3 >25.00 1 >25.00 1.00 0 NT 38-NO₂ 3 >25.00 1 >25.00 1.00 0 NT 4 5-NO₂ 3 >25.00 1 >25.00 1.00 0 NT RT= not tested.

Substitution of the nitro group was pursued for a select series (Table4), where inclusion of a nitrile group (entry 6) provided an activecompound. A fluorine atom in place of the nitro group was investigatedin tandem with other changes (Table 7, entry 8), providing an analogthat retained CPE potency. Select compounds from this set were evaluatedin a VEEV titer reduction assay. Importantly, for the compounds tested,a strong correlation was found between the CPE and titer reductionassays. A weakly potent compound in the CPE assay translated to a weaklyefficacious reduction in viral titer (Table 4, entry 5), while potentcompounds produced a robust effect in reducing viral titer (Table 4,entries 1 and 4). Surprisingly, all compounds were nontoxic (CC₅₀>25μM).

TABLE 4

VEEV TC-83 CPE assay potency mean (μm) (n = # replicates) VEEVcytotoxicity assay mean (μM) (n = # replicates) VEEV TC-83 titerreduction mean (log) IC₅₀ CC₅₀ Selectivity Entry R₁ R₂ n μM n μM(CC₅₀/IC₅₀) n log 1 NO₂ 2-F-phenyl 33 0.78 7 >50.00 >63.30 2 6.36 2 H2-F-phenyl 3 >25.00 1 >25.00 1.00 0 NT 3 CF₃ 2-F-phenyl 3 >25.001 >25.00 1.00 0 NT 4 NO₂ phenyl 18 0.25 1 >25.00 1.00 4 >7.76 5 I phenyl3 17.06 1 >25.00 >1.50 2 0.33 6 CN phenyl 3 1.14 1 >25.00 >21.90 0 NT 7SO₃H phenyl 3 >25.00 1 >25.00 1.00 0 NT 8 CO₂H phenyl 3 >25.00 1 >25.001.00 0 NT 9 NH- phenyl 3 >50.00 1 >50.00 1.00 0 NT tetrazole 0 CONH₂phenyl 3 >25.00 1 >25.00 1.00 0 NT 11 CONMe₂ phenyl 3 >25.00 1 >25.001.00 0 NT 12 2-pyridyl phenyl 3 >50.00 1 >50.00 1.00 0 NT 13 3-pyridylphenyl 3 >25.00 1 >25.00 1.00 0 NT NT = not tested.

A few other changes involving the fused phenyl ring of the quinazolinonecore included replacement of the C8 CH-atoms with a nitrogen atom or6,7-difluorosubstitution (Scheme 3). Incorporation of the nitrogen atomdid not significantly alter the CPE potency profile from that the carbonanalog at position 8 (Table 4, entry 4). Difluorosubstitution retainedCPE activity.

Given the potency afforded by the inclusion of the C6 nitrofunctionality, a SAR was investigated with this moiety preserved (Table5). Attention shifted to derivitization of the 2-fluorophenyl amidesubstituent. The N-ethyl piperazine was initially used until it wasdetermined that the N—H piperazine comparatively offered a slightlyimproved CPE potency while reducing molecular weight and adjusting cLogP(Table 5, entries 4 vs 9).

TABLE 5

VEEV CPE Assay Potency mean (μM) (n = # replicates) VEEV CytotoxicityAssay mean (μM) (n = # replicates) Selectivity VEEV titer reductionassay mean (log) Entry R₂ R₃ n IC₅₀ μM n CC₅₀ μM (CC₅₀/IC₅₀) n log 12-F-phenyl ethyl 33 0.78 7 >50.00 >63.3 2 6.36 2 3-F-phenyl ethyl 6 0.382 >25.00 >65.79 4 7.11 3 4-F-phenyl ethyl 6 0.80 2 >25.00 >31.25 4 5.884 phenyl ethyl 9 0.22 2 >25.00 >113.64 4 >7.76 5 methyl ethyl 3 >25.001 >25.00 1.00 0 NT 6 i-propyl ethyl 3 >25.00 1 >25.00 1.00 0 NT 7 benzylethyl 3 >25.00 1 >25.00 1.00 0 NT 8 H ethyl 3 >25.00 1 >25.00 1.00 0 NT9 phenyl H 3 0.15 1 >25.00 >166.67 2 >7.76 10 2-MeO- H 3 >25.00 1 >25.001.00 0 NT phenyl 11 3-MeO- H 3 5.13 1 >25.00 4.87 0 NT phenyl 12 4-MeO-H 6 0.24 2 >25.00 >104.17 0 NT phenyl 13 3-NMe₂- H 3 >25.00 1 >25.001.00 0 NT phenyl 14 4-NMe₂- H 3 >25.00 1 >25.00 1.00 0 NT phenyl 153-thiophene H 3 1.36 1 >25.00 >18.38 0 NT NT = not tested.

Compared to the 2-fluorophenyl group (Table 5, entry 1), a two-foldimprovement in CPE potency and almost one log increase in titerreduction was observed by switching to the 3-fluorophenyl group (entry2); however, removal of the fluorine atom further provided slightimprovements in both assays (entry 4) such that the titer reductionassay pegged at the maximum of >7.76 log. As noted previously, the N—Hpiperazine afforded a boost in CPE potency (entry 9) and also pegged theassay for reduction in viral titer.

Modifications to the piperazine moiety were also tested (Table 6).Replacement of the ethyl group for methyl was inconsequential, but anisopropyl group notably attenuated activity (entry 3, Table 6). Withoutbeing bound by theory, the differing activity due to the alkyl group atthis position suggests a spacial constraint in the binding site.

TABLE 6

TC-83 VEEV CPE Assay Potency mean (μM) (n = # replicates) CytotoxicityAssay mean (μM) (n = # replicates) Selectivity TC-83 VEEV titerreduction assay mean (log) (n = # replicates) Entry X R₃ n IC₅₀ μM nCC₅₀ μM (CC₅₀/IC₅₀) n log 1 CH₂ N-ethyl 33 0.78 7 >50.00 >63.30 2 6.36 2CH₂ N-methyl 9 0.79 3 >50.00 >63.30 4 7.08 3 CH₂ N-i-propyl 3 4.421 >50.00 >11.31 0 NT 4 CH₂ N-phenyl 3 >50.00 1 >50.00 1.00 0 NT 5 CH₂N—H 6 0.24 2 >25.00 >104.17 4 >7.76 6 CH₂ N-BOC 3 >25.00 1 >25.00 1.00 0NT 7 CH₂ O 3 8.67 1 >50.00 >5.77 2 0.3 8 CH₂ CH₂ 3 4.70 1 >25.00 >5.32 20.37 9 CO NH 3 >50.00 1 >50.00 1.00 0 NT NT = not tested.

An N-phenyl group (Table 6, entry 4) reduced activity. Without beingbound by theory, this may be due to steric factors or due to thepreference of a basic amine that makes beneficial interations withproximal residues in the binding site. The N—H analog was superior toall others in this set (Table 6, entry 5) for both CPE potency andreduction in viral titer. The non-basic, but still sterically-demandingN-BOC analog was inactive. Replacement of the amine with an oxygen atomor methylene unit provided activity, albeit reduced, in both CPE andtiter reduction assays (Table 6, entries 7-8, respectively). The NHamide (Table 6, entry 9) was prepared as a less spacially consuming,non-basic, NH-moiety and was determined to be inactive. Notably, thetiter reduction data was negatively impacted significantly, underscoringthe tracking between the CPE and titer reduction assays. Without beingbound by theory, these combined results suggested (but do not completelyclarify) a preference for a basic NH moiety in this region of thecompound. Surprisingly, all compounds in this set were nontoxic (>25μM).

More extensive modifications were made to the alkylpiperazine appendageto guage the need for the methylene linkage between the quinazolinonecore and piperazine group (Scheme 4). Excision of the methylene linkageand direct attachment of the piperazine to the core resulted in aninactive analog. Concomitant methylene linkage removal with nitrogenreplacement by CH provided CPE activity (3.57 μM). Reduction of theimine core was not tolerated.

Alterations in the piperazine moiety were also studied (Table 7).Migration of the NH component over one methylene unit to provide ahexahydropyrimidine (Table 7, entry 2) or ring expansion to the1,4-diazepane (Table 7, entry 3) yielded compounds that possessedrespectable CPE activity. Ring opened varieties were also investigated.A dialkyldiamine bearing a three-carbon linker showed slightly improvedpotency (Table 7, entry 4).

TABLE 7

TC-83 VEEV CPE Assay Potency mean (μM) (n = # replicates) CytotoxicityAssay mean (μM) (n = # replicates) TC-83 VEEV titer reduction assay mean(log) IC₅₀ CC₅₀ Selectivity Entry R₁ R₂ R₃ n μM n μM (CC₅₀/IC₅₀) n log 1NO₂ 2-F- phenyl

33 0.78 7 >50.00 >63.3 2 6.36 2 NO₂ phenyl

3 1.13 1 >50.00 >44.2 0 NT 3 NO₂ phenyl

3 0.94 1 >50.00 >53.2 0 NT 4 NO₂ phenyl

3 0.61 1 >50.00 >82.0 0 NT NT = not tested.

However, the greatest improvement was observed with the amidines formedfrom ring opening of the quinazolinone (Table 8, entries 1-2). Thesechanges afforded compounds with at least a 15-fold gain in CPE potencyover entry 1 of Table 7. Given the significant progress made at thispoint, other permissible or advantageous substitutions in other regionsof the scaffold were incorporated to survey effects on potency, cLogP,stability, solubility, and BBB permeability in an effort to tune andmaximize the desired profile of the potential probe. Replacement of thenitro group with either a nitrile (Table 8, entry 3) or a fluorine atom(Table 8, entry 4) did not extinguish potency; however, installation ofthe 4-methoxyphenyl substitutent at R₂ generated another valuable andpotent analog (Table 7, entry 5).

TABLE 8

TC-83 VEEV CPE Assay Potency mean (μM) (n = # replicates) CytotoxicityAssay mean (μM) (n = # replicates) TC-83 VEEV titer reduction assay meanIC₅₀ CC₅₀ Selectivity (log) Entry R₁ R₂ R₃ n μM n μM (CC₅₀/IC₅₀) n log 1NO₂ phenyl

6 0.050 2 >50.00 >1000.0 0 NT 2 NO₂ phenyl

15 0.029 10 >50.00 >1724.1 0 NT 3 CN phenyl

9 0.43 3 >50.00 >116.3 0 NT 4 F phenyl

3 6.39 1 >50.00 >7.8 0 NT 5 NO₂ 4-MeO- phenyl

15 0.039 2 >50.00 >1282 0 NT 6 NO₂ 2-F- phenyl

9 0.17 2 >50.00 >294.1 0 NT 7 CN 3-F- phenyl

3 0.84 2 >50.00 >59.5 0 NT NT = not tested

Further substitutions made towards assessing the potency of analogs areprovided in Table 9.

TABLE 9

TC-83 VEEV CPE Assay Potency mean (μM) IC₅₀ Entry R₁ R₂ R₃ X μM 1 CNphenyl

Cl 0.13 2 CN 2-F-phenyl

H 3.69 3 NO₂ 3-F-phenyl

H 0.1 4 NO₂ 4-F-phenyl

H 0.09 5 NO₂ benzyl

H 3.65 6 MeO(O)C— phenyl

H 1.98 7 NO₂

H 0.54 8 F phenyl

F 4.4 9 CN phenyl

F 0.43 10 CF₃ phenyl

H 22.3 11 NO₂ 3-MeO- phenyl

H 8.07 12 NO₂ (CH₃)₂CH—

H 27 13 H phenyl

F >300 14 NO₂ 2-MeO- phenyl

33.7

In addition, 2-((1-methylpiperidin-4-yl)amino)-5-nitro-N-phenylbenzamidewas synthesized to asses the influence of the amidine ring on thepotency.

2-((1-methylpiperidin-4-yl)amino)-5-nitro-N-phenylbenzamide

It was found that this variant exhibited a potency of 41.2 μM in theTC-83 VEEV CPE assay. Thus, structural modification of this type erodedpotency against VEEV.

While certain embodiments have been illustrated and described, a personwith ordinary skill in the art, after reading the foregoingspecification, can effect changes, substitutions of equivalents andother types of alterations to the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, prodrugs, metabolites,tautomers or racemic mixtures thereof as set forth herein. Each aspectand embodiment described above can also have included or incorporatedtherewith such variations or aspects as disclosed in regard to any orall of the other aspects and embodiments.

The present technology is also not to be limited in terms of theparticular aspects described herein, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods within thescope of the present technology, in addition to those enumerated herein,will be apparent to those skilled in the art from the foregoingdescriptions. Such modifications and variations are intended to fallwithin the scope of the appended claims. It is to be understood thatthis present technology is not limited to particular methods, reagents,compounds, compositions, labeled compounds or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. Thus, it is intended that thespecification be considered as exemplary only with the breadth, scopeand spirit of the present technology indicated only by the appendedclaims, definitions therein and any equivalents thereof.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso form part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments (for example, journals, articles and/or textbooks) referred toin this specification are herein incorporated by reference as if eachindividual publication, patent application, issued patent, or otherdocument was specifically and individually indicated to be incorporatedby reference in its entirety. Definitions that are contained in textincorporated by reference are excluded to the extent that theycontradict definitions in this disclosure.

Other embodiments are set forth in the following claims, along with thefull scope of equivalents to which such claims are entitled.

1. A compound of Formula III:

or a stereoisomer thereof and/or a pharmaceutically acceptable saltthereof, wherein: W is CH or N; X₁ is an electron withdrawing group; X₂is hydrogen or an electron withdrawing group; Y is O or S; R₁ is analkyl group, an aryl group, an aralkyl group, or a heteroaryl group; R₂is hydrogen or alkyl; R₃ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; R₄ is a hydrogen, alkyl, aryl, cycloalkyl, ornon-aromatic heterocyclyl; α is 0 or 1; and B is CH, C-alkyl, O, or N;with the provision that when B is O, R₄ is absent.
 2. The compound ofclaim 1, wherein X₁ is a halogen, a nitro group, cyano group, analkanoyl group, a carbamoyl group, an ester, a sulfonyl group, atrialkyl ammonium group, or a trifluoromethyl group.
 3. The compound ofclaim 1, wherein X₁ is a halogen, a nitro group, a cyano group, or atrifluoromethyl group.
 4. The compound of claim 1, wherein X₂ ishydrogen, a halogen, a nitro group, or a cyano group.
 5. (canceled) 6.The compound of claim 1, wherein X₂ is hydrogen.
 7. The compound ofclaim 1, wherein R₂ is hydrogen. 8-20. (canceled)
 21. The compound ofclaim 1, wherein R₁ is an alkyl group, an aralkyl group, a heteroarylgroup, or a phenyl group, wherein the phenyl group is of Formula IA:

where R₆ R₇, R₈, R₉ and R₁₀ are each independently hydrogen, halo,alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro. 22-24.(canceled)
 25. The compound of claim 1, wherein R₁ is an alkyl group, anaralkyl group, a heteroaryl group, or a phenyl group, wherein the phenylgroup is of Formula IA:

where R₆ R₇, R₈, R₉ and R₁₀ are each independently hydrogen, halo,alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro.
 26. (canceled)27. The compound of claim 1, wherein Y is O; R₁ is a heteroaryl group ora phenyl group, wherein the phenyl group is of Formula IA:

where R₆ R₇, R₈, R₉ and R₁₀ are each independently hydrogen, halo,alkoxy, alkanoyl, carbamoyl, cyano, trifluoromethyl, or nitro; R₂ ishydrogen; α is 1; R₄ is hydrogen, alkyl, aryl, cycloalkyl, non-aromaticheterocyclyl, alkanoyl, or carbamoyl; and B is CH, C-alkyl, O, or N;with the provision that when B is O, R₄ is absent.
 28. The compound ofclaim 1, wherein X₂ is hydrogen; Y is O; R₁ is an alkyl group, asubstituted or unsubstituted benzyl group, a heteroaryl group, or aphenyl group wherein the phenyl group is of Formula IA:

where R₆ is hydrogen, methoxy, halo, alkanoyl, or nitro; R₇ and R₈ areeach independently hydrogen, alkoxy, aryloxy, halo, alkanoyl, or nitro;R₉ and R₁₀ are each independently hydrogen; R₂ is hydrogen; α is 1; R₄is hydrogen, alkyl, aryl, cycloalkyl, non-aromatic heterocyclyl, oralkanoyl; and B is CH, C-alkyl, or N.
 29. The compound of claim 1,wherein X₁ is a halogen, a nitro group, a trifluoromethyl group, or acyano group; X₂ is hydrogen; Y is O; R₁ is a methyl group, an ethylgroup, a benzyl group, or a phenyl group, wherein the phenyl group is ofFormula IA:

where R₆, R₇, and R₈ are each independently hydrogen, methoxy, halo, ornitro; R₉ and R₁₀ are each independently hydrogen; R₂ is hydrogen; α is1; R₄ is hydrogen, alkyl, cycloalkyl, or non-aromatic heterocyclyl; andB is CH, C-alkyl, or N.
 30. The compound of claim 27, wherein R₆ ishydrogen.
 31. The compound of claim 27, wherein R₃ is methyl or ethyl;and R₄ is methyl.
 32. The compound of claim 27, wherein W is CH.
 33. Thecompound of claim 27, wherein R₁ is a phenyl group of Formula IA:

where R₆ is hydrogen; R₇, and R₈ are each independently hydrogen,methoxy, or halo; R₉ and R₁₀ are each independently hydrogen.
 34. Thecompound of claim 1, wherein the compound is selected from the groupconsisting of(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)-5-nitrobenzamide,(Z)-4-chloro-5-cyano-2-(1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylbenzamide,(Z)-2-((1-ethyl-4-methylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-fluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-fluorophenyl)benzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-fluorophenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenyl-5-(trifluoromethy)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(4-fluorophenyl)-5-nitrbenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(2-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-(3-methoxyphenyl)-5-nitrobenzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-isopropyl-5-nitrobenzamide,(Z)-N-benzyl-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitrobenzamide,(Z)-4-((1,4-dimethylpiperazin-2-ylidene)amino)-N-phenylpyridazine-3-carboxamide,(Z)-methyl4-((1,4-dimethylpiperazin-2-ylidene)amino)-3-(phenylcarbamoyl)benzoate,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-(thiophen-3-yl)benzamide,(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4,5-difluoro-N-phenylbenzamide,(Z)-5-cyano-2-((1,4-dimethylpiperazin-2-ylidene)amino)-4-fluoro-N-phenylbenzamide,and(Z)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-N-methyl-5-nitrobenzamide.35-36. (canceled)
 37. A composition comprising a compound of claim 1 anda pharmaceutically acceptable carrier. 38-44. (canceled)
 45. A methodcomprising: administering to a subject in need thereof an antiviraleffective amount of a compound of claim
 1. 46-53. (canceled)
 54. Acomposition comprising a compound of claim 27 and a pharmaceuticallyacceptable carrier.
 55. A composition comprising a compound of claim 33and a pharmaceutically acceptable carrier.